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Laboratory project
TRANSLATIONAL HEALTH-SCIENCE INTERNSHIP - 2019
Clinical and severity spectrum of RSV disease in hospitalized
young children in Argentina
1
Clinical and severity spectrum of RSV disease in hospitalized
young children in Argentina
Background
Lower respiratory tract illness (LRTI) is the foremost preventable cause of childhood
death and represented a major obstacle in achieving the United Nations Millennium
Development Goal to reduce global mortality in children under five (U5).1 LRTI due to
respiratory syncytial virus (RSV) is the most frequent cause of hospitalization in infants in the
world, with over three million hospital admissions every year. The disease is estimated to
cause between 66,000 and 239,000 yearly deaths in children U5.2,3 Ninety nine percent of
these fatalities occur in low income regions of middle-income countries (where 60% of deaths
in children under five occur worldwide).4
Data explaining increased severity of RSV illness in children in poor nations is limited.
Even though poverty has been postulated to influence disease outcomes through deficient
access to medical care5,6 only simple supportive interventions are available to treat RSV
disease. Therefore, it is hard to attribute the presumptive differential severity of illness
observed in rich compared to poor nations solely to the availability of medical care. In a
recently conducted study we examined RSV mortality in a low-income region from a middle-
income country where we observed that RSV is the main cause of post-neonatal infant death
in our population, affecting two different groups of infants: one at medical institutions, often
experiencing a clinically significant pneumothorax and/or sepsis, and a second group dying in
the community presumably due to RSV in association with poor access to health care.7
In children hospitalized with RSV in wealthy countries, mortality is estimated to be
approximately 1%, although it is several-fold higher in infants who are premature8,9 or who
have chronic lung disease,10 congenital heart disease,11 or primary immunodeficiency
disorders.12,13
2
Although numerous determinants of hospitalization in industrialized countries have
been described, knowledge on clinical behavior, disease complications and specific risk
factors associated with life-threatening and fatal RSV infection in vulnerable populations is
limited. We recently identified among hospitalized children novel prenatal determinants of
severe illness: alcohol and high maternal carbohydrate intake and low intake of fruits and
vegetables during pregnancy.14,15 Understanding of the contribution of social and biological
features in RSV disease severity in low-income populations is critical to define targeted
primary preventive interventions.
To contribute to the understanding of clinical and laboratory features, severity
spectrum and risk factors for severe RSV disease in low-income regions of middle-income
countries, data from a prospective active surveillance study conducted from 2011 to 2013 in
a low resource area of Argentina will be analyzed.
Specific aims
1) Characterize the demographic and clinical features of RSV disease in hospitalized
children.
2) Describe clinical outcomes and correlate viral load with RSV disease clinical
presentation, severity spectrum and risk factors for severe disease in young children.
3) Identify novel social, epidemiologic and biological risk factors for severe RSV
disease.
Methods
Study design and population. Prospective, population-based, cross-sectional, multicenter
study conducted between 2011 and 2013 in the southern Region VI of the state of Buenos
Aires in Argentina.
3
The Buenos Aires metropolitan statistical area and the neighboring city of La Plata
have an estimated population of 361,000 infants and children younger than 2 years of age. Of
these children, approximately 170,000 lack private medical insurance and receive free care at
public hospitals. In the metropolitan area, the southern region has the lowest socioeconomic
indicators, and is home to 64,600 children younger than 2 years of age without private
insurance.
Eligibility criteria.
Inclusion Criteria. Infants and young children younger than two years of age with an oxygen
saturation <93% when breathing room air, and infants <6 months of age with clinical or
radiographic evidence of pneumonia were invited to participate by pediatricians trained in
the study protocol.
Exclusion Criteria. Only patients not living in the study region were excluded from
participation.
Epidemiological & demographic variables. Upon consent by parents or legal guardians,
epidemiological data was collected by hospital investigators trained in the study protocol
using a structured questionnaire. Epidemiological data from participating children includes: I)
age (months); II) weight (upon admission and at birth); III) sex; IV) gestational age at birth; V)
mode of delivery; VI) need for oxygen supplementation or mechanical ventilation after birth
(Y/N); VII) vaccinations; VIII) congenital anomalies (intrauterine growth retardation and/or
congenital cardiopathology; Y/N); IX) breastfeeding (Y/N; exclusive or non-exclusive); X) day
care attendance (Y/N). Familial information includes: I) number of siblings < 14 years of age
living at home; II) number of people living in home; III) cigarettes habitually smoked in home
(Y/N); IV) pets at home (Y/N); V) gas or wooden stove at home; VI) presence or absence of
city water and/or sewage; VII) materials used in construction of home/floor; VIII) home in a
paved street or dirt road. Maternal obstetric history includes: I) smoking during pregnancy; II)
history of physician diagnosed asthma (Y/N); III) urinary tract infections during pregnancy
4
(Y/N); IV) alcohol consumption during pregnancy (Y/N); V) education level
(incomplete/complete primary); VI) adolescent parents (Y/N).
Clinical information. Participating pediatricians conducted a full physical examination in
every subject upon enrollment. This clinical information is presented as presence/absence of:
I) fever; II) bronchospasms; III) tachypnea; IV) tachycardia; V) wheezing; VI) retractions; VII)
vomiting; and as specific diagnoses such as VIII) bronchiolitis or IX) pneumonia. Laboratory
analyses are reported as the presence/absence of anemia and leukocytosis. Other
manifestations of severe disease include: I) death; II) admission to the floor or intensive care
unit; III) duration of hospitalization; IV) need for oxygen supplementation (Y/N; duration); V)
need for mechanical ventilation (Y/N; duration); VI) blood gas on admission when available;
VII) chest-x-ray on admission (all chest-x-rays from enrolled children were examined by two
pediatric radiologists blinded to initial readings); and specific findings from the chest x-ray:
VIII) pulmonary hyperinflation, atelectasis, and pneumonia. All participating infants and
children were followed daily until resolution of signs and symptoms by the hospital
investigators.
Sample collection and transportation. Pediatricians obtained a nasal aspirate from every
participating child. Nasal aspirates using 2 ml of sterile saline solution16 were aliquoted in four
centrifuge tubes (minimum of 0.5 ml. /each) and immediately snap frozen and stored in dry
ice. All samples were transported every other day in dry ice to the INFANT Foundation by a
professional transport service. Two tubes per patient (total of 4) were stored in two separate
-80C freezers.
Ethical considerations. The study was approved by the institutional review boards at each
participating institution, the state of Buenos Aires, and Vanderbilt University. Informed
consent was obtained from all participating parents or guardians.
5
References
1. UN. UN Millennium Development Goals Report 2015. 2015.
2. Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower respiratory
infections due to respiratory syncytial virus in young children: a systematic review and meta-
analysis. Lancet 2010; 375:1545-55.
3. Lozano R, Naghavi M, Foreman K, et al. Global and regional mortality from 235 causes
of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of
Disease Study 2010. Lancet 2012; 380:2095-128.
4. Adjagba A LJ, Yang. Sustainable Access to Vaccines in Middle-Income Countries (MICs):
A Shared Partner Strategy Report of the WHO Convened MIC Task Force. Middle Income
Country, Task Force. 2015.
5. Berman S. Epidemiology of acute respiratory infections in children of developing
countries. Reviews of infectious diseases 1991; 13 Suppl 6:S454-62.
6. Simoes EA, Peterson S, Gamatie Y, et al. Management of severely ill children at first-
level health facilities in sub-Saharan Africa when referral is difficult. Bulletin of the World
Health Organization 2003; 81:522-31.
7. Geoghegan S, Erviti A, Caballero MT, et al. Mortality due to Respiratory Syncytial
Virus: Burden and Risk Factors. American Journal of Respiratory and Critical Care Medicine
2016.
8. Cunningham CK, McMillan JA, Gross SJ. Rehospitalization for respiratory illness in
infants of less than 32 weeks' gestation. Pediatrics 1991; 88:527-32.
9. Berkovich S. Acute Respiratory Illness in the Premature Nursery Associated with
Respiratory Syncytial Virus Infections. Pediatrics 1964; 34:753-60.
10. Groothuis JR, Gutierrez KM, Lauer BA. Respiratory syncytial virus infection in children
with bronchopulmonary dysplasia. Pediatrics 1988; 82:199-203.
11. MacDonald NE, Hall CB, Suffin SC, Alexson C, Harris PJ, Manning JA. Respiratory
syncytial viral infection in infants with congenital heart disease. The New England Journal of
Medicine 1982; 307:397-400.
6
12. Fishaut M, Tubergen D, McIntosh K. Cellular response to respiratory viruses with
particular reference to children with disorders of cell-mediated immunity. The Journal of
pediatrics 1980; 96:179-86.
13. McIntosh K, Kurachek SC, Cairns LM, Burns JC, Goodspeed B. Treatment of respiratory
viral infection in an immunodeficient infant with ribavirin aerosol. American Journal of
Diseases of Children 1984; 138:305-8.
14. Libster R, Ferolla FM, Hijano DR, et al. Alcohol during pregnancy worsens acute
respiratory infections in children. Acta Paediatrica 2015; 104:e494-9.
15. Ferolla FM, Hijano DR, Acosta PL, et al. Macronutrients during pregnancy and life-
threatening respiratory syncytial virus infections in children. American Journal of Respiratory
and Critical Care Medicine 2013; 187:983-90.
16. Klein MI, Coviello S, Bauer G, et al. The impact of infection with human
metapneumovirus and other respiratory viruses in young infants and children at high risk for
severe pulmonary disease. The Journal of Infectious Diseases 2006; 193:1544-51.
7
Laboratory Project Guidelines
8
Guidelines for Lab Notebook
Everything you do in the laboratory should be in your notebook and up-to-date.
Write in ink and never erase. You can correct mistakes marking out with a single line.
What you write should be understood by other people, and you should include all the
information needed so that someone else can repeat it.
Start each new experiment on a new page. The top of the page should contain: title of the
experiment, date and page number.
Each experiment should include:
Title/purpose
Background information
Materials
Include the key materials, composition of buffers (unless they are standard or are
referenced), calculations made in preparing solutions
Pre-packaged kits: identify as to the name of the kit, the vendor, and the catalog
number.
Biological samples: identify by genus and species, strain number, tissue type,
and/or genotype with the source of the material identified. Also include date of
kit.
If any of these materials were used in previously, include only the reference to
that earlier experiment.
Procedure
Write down exactly what you are going to do before you do it.
If an experiment is a repeat of an earlier experiment, refer to the earlier
9
experiment by page or experiment number. If you make any changes, note the
changes and why.
Results
Include all your raw and analyzed data.
Conclusions/summary
Summarize all of your results and state any conclusions you can make. If the
experiment didn't work, explain what went wrong and what will you do the next
time to try to troubleshoot.
Guidelines for Journal Club Presentations
Journal club presentations will be done in groups of 2 students.
Each pair will select a paper from indexed journals. Those papers should be related to RSV
infection and epidemiology.
Each seminar aims at 30 min presentation and 15 min discussion.
Presentation need to contain the following sections:
Introduction
Methods
Results
Discussion
Conclusions
Try to use about 20 slides containing more images than words.
Focus your attention on how they arrive to conclusions from the results obtained.
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Think in what you would recommend to do or what you would be interested in testing.
Practice before. Check how much time you need. Correct the presentation if it is
necessary.
Guidelines for Final Presentations
Prepare a 1 hour presentation of the project, regarding the assigned specific aim for your
group. Presentations will be done in groups of 4 students. All students within a group
should expose in the oral presentation.
Presentation must include:
Introduction: Present clearly the context of the study and the importance of the topic.
Method: Explain the population used, the qRT-PCRs method employed, and the
statistical analysis performed. Also, include a brief explanation of how the RSV
positive control sample for qRT-PCR was obtained by tissue culture techniques. Make
it short. Try to use schemes.
Results: Expose the results from the STATA analyses that your group obtained. Use
graphics to show the results and report whether statistical significant differences are
observed.
Conclusions: Explain what you conclude and why. Discuss your results in the context
of other published studies.
It is very important to see a team work during presentation.
11
Laboratory Protocols
12
Table of Contents
Project Summary..........................................................................................................14
Cell culture....................................................................................................................15
Cell counting with Neubauer chamber.........................................................................17
RSV stock preparation and harvest...............................................................................20
Viral stock titration by immunostaining plaque assay................................................244
qPCR Practice................................................................................................................31
Statistical analysis using STATA software.....................................................................38
Cytokine quantification via ELISA..................................................................................55
Exercises....................................................................................................................... 62
Glossary of medical terms............................................................................................70
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Project Summary
14
Determination of cytokine
Determination of viral load
Cytoquine quantification
via ELISA in test
Statistical analysis of demographic, clinical, social, epidemiological and
biological variables associated with RSV
qRT-PCR of RSV positive samples using a standard
qRT-PCR of RNA samples of
Determination of
presence/absen
In vitro Cell
Stock titratio
Cell passage
RSV infectio
Virus stock
Cell culture
Objectives
❖To acquire the skills needed to work in a laminar flow cabinet under sterile conditions.
❖ To learn how to handle Vero and HEp-2 cell lines: cell passage, maintenance of cells in culture
and microscope observation.
Background
Cell passaging or splitting is a technique that enables an individual to keep cells alive
and growing under culture conditions for extended periods of time. Cells should be passed
when they are 90%-100% confluent.
Vero cells (ATCC, CCL-81) are derived from the kidney of an African green monkey,
and are one of the most commonly used mammalian continuous cell lines in microbiology,
and molecular and cell biology research.
HEp-2 (ATCC® CCL-23™) is a human epithelial cell line originally thought to be derived
from larynx carcinoma. However, recent isoenzyme analysis showed both HeLa marker
chromosomes and DNA fingerprinting, proving that HEp2 is actually a strain of HeLa cell line,
possibly established via cross-contamination during early stages of culture.
Materials
❖Growth medium: MEM 7% Fetal Bovine Serum (FBS)
❖Trypsin-EDTA
❖Phosphate Buffered Saline (PBS)
❖25 cm2 plastic flasks (T25)
❖5 ml and 10 ml plastic disposable pipettes
15
❖1.5 ml conical tubes
Protocol
1. Remove growth medium from a 100% confluent T25 monolayer of Vero or HEp-2 cells.
2. Wash cells with 2.5 ml PBS, twice.
Note: Serum produces trypsin inhibition. Therefore, it is important to rinse off any
remaining media with PBS.
3. Add 1 ml of trypsin and incubate cells at 37°C for 5 min. Gentle shaking or tapping of the
flask may help cells detach. Check at the microscope.
4. Add 4 ml of fresh MEM with 7% FBS to inactivate the trypsin.
5. Wash down cells in medium and pipette gently to break any clumps of cells.
6. Add 1 ml of cell suspension and 4 ml of growth medium to a T25 flask (5 ml final volume).
7. Incubate flasks at 37°C with 5% CO2.
8. Monitor cells daily or every other day. Change medium if necessary. When cells reach a
>90% confluent monolayer, passage cells again by repeating this protocol.
16
Cell counting with Neubauer chamber
Objective
❖ To determine the number of cells present in a cell suspension.
Background
In spite of more recent technical developments, the Neubauer chamber or
hemocytometer remains the most common method used for cell counting around the world.
The Neubauer chamber is a thick crystal slide (30 x 70 mm and 4 mm thickness), containing a
central area with two counting grids (Figure 1). Each grid is divided in nine big squares, which
are further subdivided into smaller squares (Figure 2).
Figure 1: Drawing of a Neubauer Chamber. The
Figure 2: Photograph of one counting grid of a Neubauer chamber. Squares with the
number 1 are used to count culture cells, whereas squares with the numbers 2 and 3 are
used to count white blood cells and red blood cells and platelets, respectively.
Materials
17
❖T25 Vero or HEp-2 monolayer
❖MEM 7% SFB
❖PBS
❖Trypsin-EDTA
❖Neubauer chamber
❖Trypan blue solution.
❖Micropipette with disposable tips
❖1.5 ml conical tubes
❖15 ml conical tubes
Protocol
Obtaining cell suspension:
1. Remove growth medium from a T25 monolayer of Vero or Hep-2 cells.
2. Wash cells with 2.5 ml PBS, twice.
3. Add 1 ml of trypsin and incubate cells at 37°C for 5 min. Gentle shaking or tapping of the
flask may help cells detach. Check at the microscope.
4. Add 4 ml of fresh MEM with 7% FBS to inactivate the trypsin.
5. Wash down cells in medium and pipette gently to break any clumps of cells.
6. Transfer the cell suspension to a 15 ml conical tube.
Cell counting:
7. Mix 50 µl of the cell suspension with 50 µl of trypan blue solution in a 1.5 ml conical tube.
8. Take 20 µl of the mixture and fill one of the grids of the chamber with it.
9. Using a microscope, count refringent cells in the four corner squares.
Note 1: Trypan blue is a vital dye which only stains dead cells. Live cells, which look
18
refringent at the microscope, exclude the dye.
Note 2: Choose only two edges of every small square (e.g. upper and left) to include the
cells that are on them, and respect that decision throughout the entire count, in order to
avoid double-counting the same cells.
Calculations:
10. Average the number of cells counted in the four corner squares.
11. Calculate the number of cells per ml, taking into account both the dilution of the
sample and the volume of solution that fills the chamber:
12. Calculate the total number of cells in the suspension:
19
RSV stock preparation and harvest
Objectives
❖To prepare a RSV stock.
❖To determine which MOI is the most proper to achieve a highly concentrated RSV stock.
Background
Experiments designed to demonstrate the effects of viruses require a large volume of
a virus stock with a high concentration of infectious viral particles. As viruses are obligate
intracellular parasites that infect cells in order to complete their viral cycle, a host cell line is
necessary for the replication of these pathogens. In order to obtain a proper viral stock, it is
important to establish the multiplicity of infection (MOI), which represents the ratio of the
number of virus particles to the number of target cells present in a defined space. Therefore,
to estimate a MOI it is necessary to know both the concentration of the virus and the number
of cells. The last one could be approximated by using a reference number of a full confluence
cell table (Table I), or by counting in the Neubauer chamber.
If the number of viral particles used on the infection is too low, probably the cell
culture will run out of space and die before the virus could reproduce in a high quantity. On
the other hand, if the number of viral particles is too high, all the cells are going to be
infected from the beginning and new viral particles will not have the chance to infect and
replicate. The most common values for MOI are between 0.1 to 0.01.
Most of the commonly human viruses produce characteristic cytopathic effect on one
or the other cell lines routinely used in virology laboratories. The respiratory syncytial virus
(RSV) is the major cause of lower respiratory tract infections and hospital visits during infancy
and childhood. RSV is a member of the paramyxovirus subfamily Pneumovirinae. Its name
comes from the fact that F proteins on the surface of the virus cause the cell membranes on
nearby cells to merge, forming a syncytia (multinucleated cell).
20
Table I: Growth areas and numbers of HeLa cells in various culture vessels.
Cell Culture Vessel Growth Area (cm) Number of Cells
Multiwell Plates
96-well 0.32-0.6 4.5x104
48-well 1 1.3x105
24-well 2 2.5x105
12-well 4 5.0x105
6-well 9.5 1.2x106
Dishes35 mm 8 1x106
60 mm 21 2.5x106
100 mm 56 7x106
150 mm 145 2x107
Flasks40-50 ml 25 3x106
250-300 ml 75 1x107
650-750 ml 162-175 2x107
900 ml 225 3x107
Materials
❖ RSV suspension
❖MEM 2% FBS
❖Trypsin-EDTA
❖PBS
❖70-80% confluence Vero or HEp-2 T25 flask
❖15 ml conical tubes
❖2 ml, 5 ml, 10 ml plastic disposable pipettes
❖Micropipettes with disposable tips
❖1.5 ml conical tubes
21
Protocol
1. Observe at the inverted microscope the cells grown in the T25 flask. Confluence should
be 70-80%.
2. Prepare a dilution of the viral suspension in 1 ml final volume of MEM 2% FBS. Try MOI=
0.05 and MOI= 0.01 (see example below protocol). Keep the dilution and the viral
suspension in ice.
3. Remove media from flask and wash the cells twice with 2.5 ml of PBS.
4. Infect cells by adding the 1 ml of virus dilution to the flask.
5. Incubate 60 min at 37°C, gently rock flask every 10-15 min.
6. Add 4 ml of MEM 2% FBS.
7. Incubate 72-96 h at 37°C, 5% CO2. Check the cells and the development of the cytopathic
effect (syncytia forming).
8. After incubation period, scrape to remove all cells from the flask and transfer all the
content to a sterile 15 ml conical tube. Pellet cell debris at 1500 rpm, 5 min, 4°C.
9. Separate supernatant and store it in ice; leave 1 ml to resuspend the pellet. Once the
pellet is resuspended, freeze it at -80°C for 5 minutes and then thaw it at 37ºC for 3
minutes. Repeat the freezing/thawing procedure at least 3 times.
10. Pellet cells debris at 1500 rpm, 5 min, 4°C.
11. Combine the supernatant with the one stored in ice and aliquot it to 1.5 ml conical tubes
12. Label and freeze all the tubes at -80°C.
Titrate the viral stock before any further experiment
MOI Example:
In order to prepare the 1 ml dilution of the viral suspension to infect the cell culture you need
to know the virus concentration, the number of cells and to establish the MOI.
-Virus Concentration = 3x106 PFU/ml
22
-MOI = 0.01 → It means that the infection will occur with 0.01 viral particles/cell
-Considering the table parameter of full confluence for T25 flask: 3x106 cells
100%_______ 3x106cells
80%________x = 2.4x106 cells are in the T25 flask
According to MOI:
1 cell________ 0.01 viral particles
2.4x106 cells________ x = 2.4x104 viral particles or PFU are required
Using the virus concentration:
3x106 PFU________ 1 ml
2.4x104 PFU_________ x = 0.008 ml of the virus suspension,
contained in the 1 ml of MEM 2%FBS.
23
Viral stock titration by immunostaining plaque assay
Objective
❖ To estimate the number of infectious viral particles or plaque-forming unit (PFU/ml) in the
RSV stocks.
Background
The aim of the titration is to measure the concentration of infectious virus in a
suspension. The viral titration is utilized in both research and development (R&D), in commercial
and academic laboratories as well as production situations where the quantity of virus at various
steps is an important variable.
The infectivity titers of a virus can conveniently be determined by infecting a
particular cell line, with increasing serial dilutions of the viral suspension, and determining the
number of infectious viral particles in the original viral stock by counting the number of plaques
in a monolayer of cells. This result is the plaque-forming unit (PFU/ml), which is a measure of the
number of viral particles capable of forming plaques per unit of volume.
One of the most accurate methods to visualize viral plaques in an infected cell
monolayer or tissues is the immunostaining. In direct immunostaining, an antibody that
recognizes a viral antigen is coupled directly to an indicator (a fluorescent dye or an enzyme).
Indirect immunostaining is a more sensitive method because a second antibody is coupled to
the indicator. The second antibody recognizes a common epitope on the virus-specific antibody.
Multiple second antibodies can bind to the first antibody, leading to an increased signal from the
indicator compared to direct immunostaining. To carry out this technique, virus-infected cells
are fixed to preserve cell morphology or tissue architecture. This step is usually accomplished
with acetone-methanol or paraformadehyde.
In case of using antibodies coupled to enzymes, such as Horse Radish Peroxidase
(HRP), a substrate must be added to the enzyme to reveal the position of the plaques. DAB (3,3'-
24
diaminobenzidine) in the presence of H2O2 reacts with HRP to yield an insoluble brown-colored
end product as the following reaction:
Consequently, if HRP is attached to an antibody which is bound to an antigen in a
particular area of a cell monolayer and DAB is added with H2O2, the reaction will produce an
insoluble brown DAB precipitate (DABppt) in the area where the antibody has bound the
antigen.
Materials
❖90-100% confluence Vero or HEp-2 T25 flask
❖MEM 7% FBS and 2% FBS
❖Plaque medium: MEM 2%FBS with 0,8% methylcellulose
❖Trypsin-EDTA
❖ PBS
❖ Fixing solution: acetone/methanol (1:1)
❖Antibody solution: PBS (1X) with 5% nonfat dry milk
❖Primary antibody dilution: mouse anti-RSV (R1600), dilution 1:4000 in antibody solution.
❖ Secondary antibody dilution: goat anti-mouse IgG conjugated with HRP, dilution 1:1000 in
antibody solution.
❖Substrate solution: 1 DAB pill (10 mg) in 15 ml of PBS (1X) with 40 µl of H2O2
❖24 well plates
❖1.5 and 15 ml conical tubes
❖2 ml, 5 ml, 10 ml plastic disposable pipettes
25
Protocol
DAY 1: 24-well plate preparation.
1. To prepare a 24-well plates with cells for titration start with a 90-100% confluence Vero or
HEp-2 T25 flask.
2. Remove growth medium and wash cells twice with 2.5 ml of PBS.
3. Add 1 ml of trypsin and incubate cells at 37°C for 5 min. Check at the microscope if the cells
are detached.
4. Add 4 ml of MEM 7% FBS to inactivate the trypsin.
5. Wash down cells in media, pipette gently to break up any clumps of cells.
6. Put all the cell suspension in a 15 ml conical tube.
7. Take 50 µl of the cell suspension and mix it with 50 µl of Trypan blue.
8. Estimate the initial concentration of cell suspension by counting in the Neubauer chamber.
9. Calculate the initial volume necessary in order to have a final concentration of 2x105
cells/ml in the cell suspension (final volume of suspension needed is 12 ml). Use the following
formula:
Ci x Vi = Cf x Vf
Ci = Initial concentration of cell suspension (estimated with Neubauer Chamber).
Vi = Initial volume (X of the equation, volume you need to pipette from the initial cell
suspension)
Cf = Final concentration of cell suspension: 2x105 cells/ml.
Vf= Final volume: 12 ml (volume needed to prepare a 24-well plate).
10. Pipette Vi of initial cell suspension and add the necessary volume of MEM 7% FBS in order to
reach Vf.
11. Add 0.5 ml of cell suspension to each well of the 24-well plate.
26
12. Incubate the plate 24 h at 37°C, 5% CO2.
DAY 2: Virus titration by plaque assay
1. Observe the 24-well plates in the microscope to assess the required cell confluence
(approximately 70-80%).
2. Prepare the viral stock dilutions in MEM 2% FBS. Add 50 µl of viral stock to 450 µl of MEM 2%
SFB to obtain the 1/10 (-1) dilution. Repeat the procedure by adding 50 µl of (-1) dilution to
450 µl of MEM 2% SFB to obtain the 1/100 (-2) dilution. Repeat again to complete the
scheme shown below. Keep the viral dilutions in ice!!
3.
3. Label the 24-well plate as shown in the next page. The CC wells are the controls, cells with no
viral infection.
27
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MEM 2% FBS
4. Remove growth medium.
5. Add 200 µl of the appropriate dilution to each well (two replicates per dilution).
6. Incubate 60 min at 37°C, gently rock the plate every 10-15 min.
7. Add 1 ml of plaque medium to all wells.
8. Incubate cells 3-4 days at 37°C, 5% CO2.
DAY 3: Plaque assay / Fixation
1. Remove plaque medium by invertion of the plate.
2. Add 1 ml of fixing solution per well.
3. Incubate the plate at 4°C for 60 min.
4. Remove the fixing solution. At this point the plate can be frozen at -20°C to be stained later.
DAY 4: Plaque assay / Immunostaining
1. Thaw the plate 30 min at 37°C before using it.
2. Wash the plate twice with 0.5 ml PBS per well.
3. Add 200 µl of the primary antibody dilution to each well.
4. Incubate the plate at 37°C for 60 min.
5. Remove the primary antibody dilution and wash twice with 0.5 ml PBS per well.
6. Add 200 µl of the secondary antibody dilution to each well.
7. Incubate the plate at 37°C for 60 min.
8. Remove the secondary antibody dilution and wash twice with 0.5 ml PBS per well.
9. Add 200 µl of the substrate solution to each well.
10. Incubate the plate for 10-15 min at room temperature. Protect from light!!
11. Remove the substrate solution and stop the reaction with 1 ml of distilled water per well.
Discard.
12. Air-dry the plates.
13. Estimate the virus titer as Plaque Forming Units per ml (PFU/ml) by counting the number of
plaques at an appropriate dilution. Use the following formula:
28
Virus titer (PFU/ml) = (Average number of PFU) / (Dilution x 0.2 ml)
e.g:
If you count 54 and 62 PFU (average 58) in ‘‘-3 wells’’ (dilution =1/1000 or 10-3), the
virus titer would be:
(PFU/ml) = 58 PFU / (10-3 x 0.2 ml) = 2.9 x 105 PFU/ml
29
30
Quantitative Real-Time Polymerase Chain Reaction Objetives:
Learn qPCR basics Detect RSV using a molecular biology technique Use qPCR to quantify RSV
RNA extraction:
RNA was extracted using The MagMAX RNA Isolation Kit, designed for rapid high throughput purification of total RNA in 96-well plates for semi-automated isolation on the MagMAX Express-96 Magnetic Particle Processor (Life Technologies). This technique was used to extract RNA from nasal washes.
For more information visit: www.thermofisher.com
38
qPCR technique Basics
39
qPCR Components & Steps: Overview
40
Graphics
41
ProtocolsViral Detection
Prepare the qPCR reaction mix
1) Prepare in a tube the master mix with the following reagents & quantities:
μl/well μl/plate2X RT-PCR Buffer 5 510
25X RT-PCR Enzyme Mix 0,4 40,8RSV F-primer 0,3 30,6RSV R-primer 0,3 30,6
RSV Probe 0,1875 19,125Water 1,9625 200,175
Sample 2
2) Load the plate
a) Transfer 8 μL of PCR reaction mix into each well of a 96 well reaction plate.b) Add 2 uL of the sample into each wellc) Seal the plate with the plate-cover. d) Load the plate into the instrument
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Instrument: StepOne plus. For more information visit: https://www.thermofisher.com/
3) Run the plate following the next parameters:
4) Data analysis
Quantification
Once the viral detection results are available, we will proceed to choose those RSV positive samples for viral quantification.
Viral quantification will be determined by the number of amplification cycles needed for a positive PCR test (cycle threshold, CT). Previous studies have shown a highly significant inverse linear relationship between viral load and CT-values. For reference, we will run a standard curve using the RNA extracted from viral RSV stocks (from known titers) and we will prepare a 10-fold serial dilutions (from -1 to -7).
1) Prepare the standard serial dilutions in a plastic tube.
a) Add 18 ul of water in each tube.
b) Add 2 ul of RSV RNA to the -1 tube. Mix well. Continue with the serial dilutions as shown above.
43
Prepare the qPCR reaction mix:
1) Prepare in a tube the master mix with the following reagents & quantities:
μl/well μl/plate2X RT-PCR Buffer 5 510
25X RT-PCR Enzyme Mix 0,4 40,8RSV F-primer 0,3 30,6RSV R-primer 0,3 30,6
RSV Probe 0,1875 19,125Water 1,9625 200,175
Sample 2
2) Load the plate
a) Transfer 8 μL of PCR reaction mix into each well of a 96 well reaction plate.b) Add 2 uL of the sample or standar into each well. See above the standard curve distribution.c) Seal the plate with the plate-cover. d) Load the plate into the instrument.
3) Run the plate (same conditions as those used for viral detection).
4) Data analysis.
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Statistical analysis using STATA software
Objectives
Learn basic commands on STATA statistical software
Use STATA to analyze the descriptive statistics and perform regression analysis of the
relevant variables in the project database.
Background
We will be working with three file types: STATA datasets (.dta), do files (.do) and logs
(.smcl). The dataset stores variables and observations. The do file is a list of commands that
you can edit, save and re-execute. The log is a history of all the commands, failed commands
and edits made in the dataset. All can be accessed from the file menu.
STATA user interface
45
Write your commands here
Results appear here
History of commands
List of variables
Basic commands
list Lists all variables list var Lists specified variablelist var if ….
generate Generates new variablegen int varname… Generates new numeric variable
replace Replace specified observationscount
count if… Counts specified observations sum Basic statistics for quantitative variables
sum vartabulate For categorical variables
tab var Frequencies and percentages of specified variables outcomes
tab var if… Frequencies and percentages of specified variables outcomes that meet if-clause criteria
tab var1 var2 Contingency tabletab var1 var2, row column all Chi2 test
ranksumranksum quantitativevar, by (categoricalvar )
Mann-Whitney ranksum test
logistic Dep variable must be categoricallogistic depvar indepvar1 Logistic regression- Odds ratios
helphelp command Help using specified command
Basic operators
== Equal to!= Not equal to& and| or><etc As expected. No data
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Examples:
Descriptive Statistics:
Categorical variables: use the tabulate command to generate a frequency table.
Of 317 total patients, 141 (44.5%) are female and 176 (55.5) are male.
Quantitative variables: use the summarize command for basic descriptive statistics.
The 317 patients in the study population have a mean age of 7.1 (±5.7) months,
ranging from 0 to 24 months old.
To describe groups within variables, use an if clause.
The mean age of the male patients included in this study was 7.1 (±5.5) months.
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Mann-Whitney ranksum test: comparing one categorical variable and one quantitative variable
The Mann-Whitney test is for data for which we cannot assume a normal distribution.
The null hypothesis is that the two groups have the same distribution. This is a rank sum test
that assigns a rank to each observation regardless of its group and then compares the sums
of the ranks of each group. These sums are compared to the expected sums of the null
hypothesis.
Let´s consider a quantitative variable, weeks of gestation, and a categorical variable,
admittance to the neonatal intensive care unit (NICU). To compare the mean number of
weeks gestation of infants who were or were not admitted to the NICU in this study we will
use the command ranksum:
The result we are interested in is the p-value, which in this case is less than 0.05. Stata
only reports four decimal places for p-values; we can report this value as <0.0001. We can
48
reject the hypothesis that the mean gestational weeks of infants admitted to the NICU is
equal to the mean gestational weeks of infants that were not admitted.
To calculate the respective mean gestational weeks for the patients who were
admitted/ not admitted to the NICU we return to the summarize command.
We can report that infants admitted to the NICU had significantly fewer weeks of
gestation (35.7± 3.6) than infants who were not admitted (39.0 ± 1.5) (p<.0001).
Creating variables:
To create a categorical variable in which premature infants are distinguished from full
term infants we will use the commands generate and replace:
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50
Chi 2 Test: Comparing two categorical variables with a two-way frequency table
Using our new dichotomic variable preterm, we can analyze the data using a Chi2 test
of independence. The null hypothesis is that the distributions of the two variables are
independent. The result is based on comparing the distribution of the data to the expected
distribution of the null hypothesis.
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We are interested in the statistic for Pearsons Chi2, which is a test of independence.
Since the p-value is less than 0.05, we can reject the null hypothesis and report that the
proportion of preterm infants admitted to the NICU is significantly larger than the proportion
of full term infants admitted to the NICU in this patient population (73.2 % vs. 8.3%)
(p<0.0001).
Univariate logistic regression : reporting Odds Ratios
Odds ratios report the probability of an outcome given exposure to a secondary
variable. It is the ratio of the odds that the outcome will occur in the event of exposure to the
odds that the outcome will occur in the absence of exposure.
P (exposure )1−P (exposure )
Therefore,
OR=1 Exposure to the secondary variable does not affect the odds of outcome OR>1 Exposure to the secondary variable is associated with higher odds of outcome OR<1 Exposure to the secondary variable is associated with lower odds of outcome
Let’s consider the odds of being admitted to the neonatal intensive care unit for preterm
infants compared to full term infants.
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The odds ratio is 30, and the 95% confidence interval is 13.3-67.6. Since the OR is not
equal to 1 and the 95% CI does not include 1, we can exclude the possibility that the exposure
(being born after fewer than 37 weeks gestation) has no effect on the odds of the outcome
(NICU admission).
We can report that in this study population, the odds of a preterm infant being
admitted to the NICU were 30 times higher than for full term infants.
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Specific Objectives
Group 1. Characterize the demographic and clinical features of RSV disease in
hospitalized children
Patient population subgroups: Children under 2 years of age with respiratory illness
who are PCR positive for RSV vs. Children under 2 years of age with respiratory illness who
are negative for RSV.
Variables of interest:
A. Are the patients who (are) (variables a-j) significantly more likely be infected with
RSV?
a. Very young
b. Underweight
c. Male
d. Premature
e. Born via c-section
f. Required O2 at birth
g. Vaccinated
h. Have congenital anomalies
i. Breastfeed
j. Attend day care
B. Are RSV+ patients significantly more likely to exhibit any of these signs and symptoms
of disease than the RSV negative patients?
a. Fever
b. Bronchospasms
c. Tachypnea
d. Tachycardia
e. Wheezing
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f. Retractions
g. Vomiting
h. Bronchiolitis or pneumonia
i. Anemia
j. Leukocytosis
k. Death
l. Admission to the floor or intensive care unit
m. Duration of hospitalization
n. Need for oxygen supplementation and mechanical ventilation during
hospitalization
o. Oxygen saturation on admission
p. Pulmonary hyperinflation
q. Atelectasis
r. Pneumonia
C.
Identify categorical and quantitative variables to choose appropriate statistical tests
for analysis
Generate the dichotomic variable RSVpos in which patients with a positive result are 1
and patients with a negative result are 0.
Commands for quantitative variables
Do Mann-Whitney tests comparing the distribution of data for each quantitative
variable by RSV positive and negative patients.
e.g. ranksum weightg, by (RSVpos)
Follow-up Mann-Whitney tests with descriptive statistics of the quantitative variable.
e.g. sum weightg if RSVpos ==0
sum weightg if RSVpos ==1
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Report significant and interesting non-significant results.
Commands for categorical variables
Do Chi2 tests with RSVpos and your variables of interest.
e.g. tab RSVpos fever, row column all
Where appropriate (significant Chi2) follow up with logistic regression.
e.g. logistic RSVpos rx_pulmhyperinf
logistic RSVpos fever
Report significant and interesting non-significant results.
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Group 2: Describe clinical outcomes and correlate viral load with RSV disease
clinical presentation, severity spectrum and risk factors for severe disease in
young children
Patient population subgroups: Children under 2 years of age with non-severe
respiratory illness and a positive PCR result for RSV vs. Children under 2 years of age with
SEVERE respiratory illness and a positive PCR result for RSV (We will define severe respiratory
illness as oxygen saturation upon admission of less than or equal to 87 %).
Variables of interest:
A. Which of the following signs and symptoms are characteristic in infants with non-
severe RSV vs. severe RSV?
a. Fever
b. Bronchospasms
c. Tachypnea
d. Tachycardia
e. Wheezing
f. Retractions
g. Vomiting
h. Bronchiolitis
i. Anemia
j. Leukocytosis
k. Pulmonary hyperinflation
l. Atelectasis
m. Pneumonia
B. Is viral load correlated with worse outcomes?
a. Severity
b. Admission to the floor or intensive care unit,
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c. Need for oxygen supplementation and mechanical ventilation during
hospitalization
d. Death
C. Is viral load correlated with a particular manifestation of disease?
a. Fever
b. Bronchospasms
c. Tachypnea
d. Tachycardia
e. Wheezing
f. Retractions
g. Vomiting
h. Bronchiolitis
i. Anemia
j. Leukocytosis
k. Pulmonary hyperinflation
l. Atelectasis
m. Pneumonia
Identify categorical and quantitative variables to choose appropriate statistical tests
for analysis
Generate the dichotomic variable RSVpos in which patients with a positive result
are 1 and patients with a negative result are 0.
Generate new dichotomic variable defining severe RSV as: 1 if patients have
positive RSV pcr and an oxygen saturation upon admission of less than or equal to
87; and 0 if patients have positive RSV pcr and an oxygen saturation upon
admission above 87.
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Commands for categorical variables
Do Chi2 tests with RSVsev and your variables of interest.
e.g. tab RSVsev nicu, row column all
Where appropriate (significant Chi 2) follow up with logistic regression.
e.g. logistic RSVsev nicu
logistic RSVsev wheezing
Report significant and interesting non-significant results.
Commands for quantitative variables
Do Mann-Whitney tests comparing the distribution of RSV quantity for each
categorical variable of interest.
e.g. ranksum RSVload, by (nicu)
Follow-up Mann-Whitney tests with descriptive statistics of the quantitative
variable.
e.g. sum RSVload if nicu ==0
sum RSVload if nicu ==1
Report significant and interesting non-significant results.
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Group 3: Identify novel social, epidemiologic and biological risk factors for
severe RSV disease
Patient population subgroups: Children under 2 years of age with non-severe
respiratory illness and a positive PCR result for RSV vs. Children under 2 years of age with
severe respiratory illness and a positive PCR result for RSV (We will define severe respiratory
illness as oxygen saturation upon admission of less than or equal to 87 %).
Variables of interest:
A. Do any of the following factors represent a significantly increased risk of severe
disease?
a. Age
b. Weight
c. Sex
d. Gestational age
e. Type of birth
f. Requirement for o2 at birth
g. Vaccination
h. Congenital anomalies
i. Intrauterine growth retardation
j. Breastfeeding
k. Daycareattendence
l. Number of siblings
m. Number of people living in home
n. Cigarettes habitually smoked in home
o. Pets at home type of stove at home
p. Type of stove at home
q. Presence or absence of city water and/or sewage
r. Materials used in construction of home/floor;
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s. Home in a paved street or dirt road.
t. Mother smoking duringpregnancy;
u. Mother has asthma
v. Mother had urinary tract infections during pregnancy
w. Mother consumed alcohol during pregnancy
x. Mother has finished elementary school
y. One or both parents are adolescents
Identify categorical and quantitative variables to choose appropriate statistical tests
for analysis
Generate the dichotomic variable RSVpos in which patients with a positive result
are 1 and patients with a negative result are 0.
Generate new dichotomic variable defining severe RSV as: 1 if patients have
positive RSV pcr and an oxygen saturation upon admission of less than or equal to
87; and 0 if patients have positive RSV pcr and an oxygen saturation upon
admission above 87.
Commands for categorical variables
Do Chi2 tests with RSVsev and your variables of interest.
e.g. tab RSVsev pets, row column all
Where appropriate (significant Chi2) follow up with logistic regression.
e.g. logistic RSVsev pets
Report significant and interesting non-significant results.
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Commands for quantitative variables
Do Mann-Whitney tests comparing the distribution of your quantitative variables
of interest by RSV severity.
e.g. ranksum weightg, by (RSVsev)
Follow-up Mann-Whitney tests with descriptive statistics of the quantitative
variable.
e.g. sum weightg if RSVpos ==0
sum weightg if RSVpos ==1
Report significant and interesting non-significant results.
Bibliography /Other resources
The STATA help command.
McDonald, J.H. 2014. Handbook of Biological Statistics (3rd ed.). Sparky House
Publishing, Baltimore, Maryland. Online version: http://www.biostathandbook.com
Szumilas, M. (2010). Explaining Odds Ratios. Journal of the Canadian Academy of Child
and Adolescent Psychiatry, 19(3), 227–229.
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Cytokine quantification via ELISA
Objective
❖To determine the concentrations of IL-1β, IL-9 and IFN-γ in the samples provided.
Background
ELISA (Enzyme-Linked ImmunoSorbent Assay) is an immunochemical test involving an
enzyme and an antibody to detect a specific target molecule. ELISA combines the specificity
of antibodies with the sensitivity of enzyme assays, by using immunoglobulin conjugated to
an easily assayed enzyme. ELISA tests are useful to detect substances that have antigenic
properties, primarily proteins, in complex mixtures. Some of these include hormones,
bacterial antigens and other antibodies.
There are different variations of ELISA, one of the most highly efficient, in sample
antigen detection, is the Sandwich ELISA (Figure 1) which measures the amount of antigen
between two layers of antibodies. The antigens to be measured must contain at least two
antigenic sites capable of binding the antibodies, since at least two antibodies act in the
sandwich. To perform this assay, one antibody (the “capture” antibody) is purified and bound
to a solid phase typically attached to the bottom of a plate well. Antigen is then added and
allowed to interact with the capture antibody. Unbound products are then removed with a
wash and a second antibody (the “detection” antibody) binds the remaining antigen, thus
completing the “sandwich”. The detection antibody is linked to an enzyme capable of
converting a specific chromogenic substrate to a detectable signal.
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Figure 1: Sandwich ELISA in which detection antibody is conjugated to HRP (HRP:
Horseradish peroxidase, TMB substrate: Tetramethylbenzidine).
The sandwich ELISA kit Ready-Set-Go! uses a biotinylated detection antibody to
increase the sensitivity of the assay. In this common variation, incubation with the detection
antibody is followed by incubation with Avidin or another biotin binding molecule which is
conjugated with multiple molecules of HRP, the catalyzing enzyme (Figure2). The presence of
multiple enzymes for each antigen increases assay sensitivity.
Figure2. Sandwich ELISA in which the detection enzyme is linked to the immunocomplex by biotin-streptavidin
interactions (HRP: Horseradish peroxidase, TMB substrate: Tetramethylbenzidine).
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Materials:
Capture Antibody: Pre-titrated, purified anti-human IL-1β or IL-9 or IFN-γ monoclonal
antibody, 1 vial (500 μl) Capture Antibody Concentrate (250 x)
Detection Antibody: Pre-titrated, biotin-conjugated anti-human IL-1β or IL-9 or IFN-γ
monoclonal antibody , 1 vial (500 μl) Detection Antibody Concentrate (250x)
Standard: Recombinant human IL-1 or IL-9 or IFN-γ protein for generating standard
curve and calibrating samples, (lyophilized)
Coating Buffer: 1 vial (12 ml) 10x
Assay Diluent: (150 ml) 5x
Detection enzyme: Pre-titrated Avidin-HRP , 1 vial (500 μl)
Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution, (100 ml)
Plastic reservoirs.
Other materials needed:
Wash Buffer: 1x PBS, 0.05% Tween-20
Stop Solution: 2N H2SO4
Pipettes and pipettors
Refrigerator
96-well plate (Corning Costar 9018)
Reagent preparation:
Coating Buffer (1x)
Make a 1:10 dilution of PBS (10x) in deionized water.
Assay Diluent (1x)
Make a 1:5 dilution of Assay Diluent (5x) in deionized water.
Capture Antibody
Dilute 48 µl capture antibody (250x) in 12 ml Coating Buffer (1x).
Standard
Preparation of the top standard dilution:
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IL-9: Reconstitute standard to a final concentration of 100 pg/ml in deionized water.
IFN- γ : Reconstitute standard to a final concentration of 500 pg/ml in assay diluent.
IL-1 β : Reconstitute standard to a final concentration of 150 pg/ml deionized water.
Detection Antibody
Dilute 48 µl detection antibody (250x) in 12 ml Assay diluent (1x).
Avidin-HRP
Dilute 48 µl avidin-HRP (250x) in 12 ml Assay diluent (1x).
Experimental procedure
Day 1
1. Coat Corning Costar 9018 ELISA plate with 100 μl/well of capture antibody in Coating
Buffer (dilute as noted in point 1 of Reagent Preparation). Seal the plate and incubate
overnight at 4°C.
Day 2
2. Prepare Assay Diluent.
3. Aspirate wells and wash five times with 200 μl/well of Wash Buffer. Allowing time for
soaking (~1 minute) during each wash step increases the effectiveness of the washes. Blot
plate on absorbent paper to remove any residual buffer.
4. Block wells with 200 μl/well of Assay Diluent. Incubate at room temperature for 1 hour.
5. Prepare top standard concentration.
6. Aspirate/wash as in step 3. Repeat for a total of 5 washes.
7. Perform 2-fold serial dilutions of the standards with Assay Diluent to make the standard
curve:
Add 100 μl of Assay Diluent to all standard wells. Add 100 μl reconstituted standard in
duplicate into well A1 and A2. Mix the contents of wells A1 and A2 by repeated aspiration
and ejection and transfer 100 μl to wells B1 and B2, respectively. Take care not to scratch
surface of the microwells. Repeat procedure 5 times.
8. Add 100 μl/well of prediluted samples to their corresponding wells.
9. Add 100 μl/well of Assay Diluent to the blank wells.
66
10. Seal plate and incubate at room temperature for 2 h at room temperature or overnight at
4°C.
Day 3
11. Aspirate/wash as in step 3. Repeat for a total of 5 washes.
12. Prepare Detection Antibody (see point 6 of Reagent Preparation)
13. Add 100 μl/well of diluted Detection Antibody to all wells. Incubate at RT for 1 hour.
14. Aspirate/wash as in step 3. Repeat for a total of 5 washes.
15. Prepare Avidin-HRP (see point 6 of Reagent Preparation)
16. Add 100 μl/well of diluted Avidin-HRP to all wells. Incubate at RT for 30 min.
17. Aspirate/wash as in step 3. Repeat for a total of 7 washes.
18. Add 100 μl/well of Substrate Solution to each well. Incubate plate at room temperature
for approximately 15 minutes.
19. Add 50 μl of Stop Solution to each well.
20. Read plate at 450 nm. If wavelength substraction is available, substract the values of 570
nm from those of 450 nm and analyze data.
Calculations
Average the duplicate readings for each standard, blanks and samples. Subtract the
average of the blank to all the data.
To create the standard curve the data of the standards readings must be linearized by
plotting the log of the concentrations versus the absorbance. Find the line that fits the
best with regression analysis.
Calculate the concentration of the samples by replacing the absorbance readings in the
equation of the standard curve.
Concentrations from the samples must be multiplied by the dilution factor if necessary.
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68
69
Exercises
THEORETICAL QUESTIONS
• What are the differences between viruses, prokaryotes and eukaryotes?
• What bonds does trypsin break? What is trypsin? What is the role of EDTA in the
trypsin preparation?
• What is the purpose of adding fetal bovine serum to medium used for cell cultures?
• Why are cancerous higher eukaryote cells used for viral studies?
CELL COUNTING
1. You have prepared four T25 flasks containing 70% confluent monolayers of Vero cells.
After resuspending them in 5 ml per flask, you mixed the four cell suspensions and took a
sample of 50 l. You then added 50 l of trypan blue solution and filled one grid of a
Neubauer chamber. The figure shows what you observed under the microscope.
70
a. Why some cells are stained blue while others are refringent?
b. Calculate the number of live cells per milliliter.
c. Calculate the total number of live cells you obtained.
d. Calculate the % viability.
e. If you need 2 x 106 viable cells, how much cell suspension you should take?
f. If you need 2 x 106 viable cells in a concentration of 100,000 cells/ml, what can you
do?
2. A HEp-2 cell culture has been resuspended in 10 ml. You diluted 100 l of cell suspension
with 100 l of trypan blue and loaded 10 l on your Neubauer chamber. After counting, the
mean count for refringent cells was 35 cells.
a. What is the dilution factor of your stained cells?
b. How many live cells are in your 10 ml suspension?
3. A volume of 50 l from a 3 ml resuspended cell culture was mixed with 50 l of trypan
blue. Then, 10 l of the recently prepared 100 l cell suspension were used to load a
Neubauer chamber. This is what you observed under the microscope:
71
Count, average, and inform viable cell concentration in the 3 ml cell suspension.
4. A Vero cell culture has been recently resuspended and has been diluted by 1/6. This
dilution was used to make serial dilutions 1/10. You take 100 l of each dilution and stain
with 100 l of trypan blue. Your Neubauer chamber reading is the following:
dilution 10-6 dilution 10-5 dilution 10-4
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Which dilution do you use for counting? Why? Having chosen the dilution with which
counting shall be done, calculate how many cells are present in said dilution and thereafter
calculate the amount in the original cell suspension.
MULTIPLICITY OF INFECTION (MOI)
1. What is the MOI used in the following paper?
Jonathan M. Ciencewicki, et. al. A genetic model of differential susceptibility to human
respiratory syncytial virus (RSV) infection. doi: 10.1096/fj.13-239855. PMC3963017.
What does it mean?
2. Calculate the amount of ul of viral suspension needed for MOI = 1, having 5x103 viral
particles/l and 1x104 cells in a 12 well plate.
3. If 2x106 cells are infected by 0.5 ml of virus with a titer of 1.1 x 104 PFU/ml, inform the
multiplicity of infection.
4. You need to prepare 2 ml of viral suspension to infect HEp-2 cells grown in two T75 flasks.
The viral stock has a concentration of 2.5 x 106 PFU/ml.
You need to infect with 1 viral particle each 20 cells, so the MOI you need is ______________.
Considering the table parameter of full confluence for T75 flasks, it would have
____________ cells/T75. If you have 75% confluent T75 HEp-2 cells, there are
_______________ cells/T75. So, in total you have _______________ cells.
According to MOI: 1 cell ------------ _________ viral particles
a. Calculate the number of viral particles you need.
73
b. Calculate the volume of viral stock suspension you need.
c. To reach 2 ml of viral suspension, how much MEM should you add?
5. Instead of having a successful infection when doing the previous question experiment,
most of the cells died and the infection was a failure.
a. Knowing your cells were healthy and had a 90% confluence, what could have been the
reason for cell death?
b. What can you do? Show two different conditions you would like to try each in one
T25, using the same viral stock.
6. You take 16.9 l of a virus suspension and add it to 983.1 l of MEM containing 2% FBS.
a. What is the dilution factor? Thereafter you inoculate the whole volume of your viral
dilution in a T25 flask with HEp-2 cells, that have reached 100% confluency. You have
3 x 104 PFU/ml in the T25 flask.
b. What was the concentration of the initial viral stock? What was its MOI?
SERIAL DILUTIONS
1. You are going to titrate a virus stock you prepared, and you want to try the next dilutions
of your stock: 1/10, 1/100, 1/500, 1/1000, and 1/5000. You decided to do triplicates using
200 l in each.
Explain the procedure you are going to follow in order to prepare the dilutions. Remember
you need some extra ul of each dilution in order to pipette properly. Use a scheme to make it
clear.
74
2. A colleague was trying to titrate another viral stock using a serie of dilutions from 1/5 to
1/1000. He couldn’t count PFU in any well because there were too many PFU per well. Which
dilutions would you recommend him to prepare? Show him a scheme of the procedure you
would use to prepare those.
3. In BASIC PROTOCOL 4 of the following article:
MinKyung Yi. Hepatitis C Virus: Propagation, Quantification, and Storage. doi:
10.1002/9780471729259.mc15d01s19. PMC3039288
Read point 2 (“Prepare…”). Why do they do that? How would you prepare those dilutions?
Pay attention to know how many wells you will use (point 1), counting a negative control
(without virus). Also, consider the volume you will need of each (point 3).
CALCULATION OF VIRAL TITER
1. Observe the following 24-well plate.
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a. What are the differences between the first 2 rows and the second 2 rows? How can
you explain this?
b. Which column and rows would be used to calculate the PFU/m in this titration plate?
c. After determining the correct column, and knowing that the row “dilution”was made
making serial dilutions of 1/10 and the volume of inoculum was 200 l/well, inform
the PFU/ml of the viral stock.
2. You are titrating a viral stock which date is 17/3/2018. Suppose the following photo is
representative of an average.
Estimate the virus titer as PFU/ml by counting the number of plaques. The virus dilution is
1/2000 and the volume of inoculum is 200 l/well.
3. The following is a representative titration photo of a different viral stock, date 3/4/2018.
The virus dilution is 1/500 and the volume of inoculum is 200 l/well. Which viral stock has a
higher virus titer, this one or the stock date 17/3/2018?
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GLOSSARY OF MEDICAL TERMS
Atelectasis: partial collapse or incomplete inflation of the lung
Bronchospasms: constriction of bronchi and bronchioles
Bronchiolitis: acute inflammatory injury of the bronchioles that is usually caused by a
viral infection
Leukocytosis: increase in the total number of white blood cells
O2 saturation: percent of hemoglobin binding sites in the blood that are carrying oxygen
Pneumonia: infection that inflames the air sacs in one or both lungs
Tachycardia: heart rate of more than 100 beats per minute
Tachypnea: abnormally rapid breathing
Wheezing: high-pitched whistling sound made while breathing
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