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Flow Cytometry 101:How Flow Cytometry Can Help Advance Your Research
Components of Flow Cytometry
• Fluidics• Lasers• Optics• Data Analysis• Fluorescence- activated Cell Sorting
Fluidics System Transports Cells
Laser
Hydrodynamic Focusing
SheathFluid
LightScatter
Fluorescent Signal
Sample
Sample Flow Rate Impacts Alignment
High Sample Flow Rate =Small difference = Larger Core
Low Sample Flow Rate=Large difference = Smaller Core
Lasers are ideal for flow cytometry
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Light Amplification byStimulatedEmission ofRadiation
1. Stable2. Focused3. Single Wavelength
Lasers Excite Fluorophores
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Optics Detect Emission of Fluorescence
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•Dichroic Mirrors•Filters•Detectors
Multiparametric analysis is possible
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Sample Preparation
• Incubate with Antibody• Wash to remove unbound Antibody • Analyze
Fluorescence-Activated Cell Sorting
Laser
Hydrodynamic Focusing
SheathFluid
LightScatter
Fluorescent Signal
Sample
++
+
--
--
waste
How can I use Flow Cytometry?
• Immunophenotyping• Intracellular proteins
• Transcription Factors• Phosphorylation Status
• Extracellular proteins• Receptor Density
• Viability/Apoptosis• Caspase activation• Annexin-V presentation• TUNEL
• Proliferation• CFSE• Cell Cycle Analysis
• Isolation/Sorting
Case Study – Immunophenotyping
Request:Characterize AML samples for expression of specific markers so customer could purchase samples of interest
Approach:Multiparametric analysis of CD47, CD147, CD9 and proprietary protein
Results:Identified specific patient samples that could be used in downstream analysis for customer
Case Study – Pathway Activation
Request:Treat MM samples with proprietary compound and determine phosphorylation status of protein
Approach:After treatment, fix and permeabilize cells and quantify phosphorylation of protein in cell subpopulations
Results:Compound inhibited phosphorylation of proteins associated with proliferation/ apoptosis in CD38- and CD138-expressing cells
CD38 CD1380
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Case Study – Apoptosis
Request:Treat CLL samples with proprietary compound and determine apoptotic affect
Approach:After treatment, quantify apoptosis and necrosis using Annexin-V and 7-AAD
Results:Compound induced apoptosis/necrosis in CLL samples
Case Study – Receptor Density
Request:Determine receptor density of specific antigens in AML patients
Approach:Measure Mean Fluorescent Intensity (MFI) of antibodies compared with beads bound with absolute levels of IgG
Results:Quantified antigen binding capacity in multiple AML samples
Case Study – Multiparametric Sorting
Request:Isolate CD14+CD16+ and CD14+CD16- subpopulations from patient with RA undergoing treatment with proprietary compound
Approach:Samples sent from doctor at regular intervals were sorted based on CD14- and CD16-expression for RNA extraction and transcriptional analysis.
Results:CD14+CD16+ and CD14+CD16- fractions were isolated from fresh PB MNC.
Case Study –Sorting based on MFI
Request:Sort CD56-bright and CD56-dim populations for downstream analysis
Approach:Isolate CD56+ cells using immunomagnetic isolation and sort CD56 subpopulations based on MFI
Results:Isolated CD56-expressing cells based on relative expression level for RNA analysis
Flow Cytometry is a powerful research tool
Cell Isolation
Proliferation
Apoptosis (Annexin V/PI)Pathway Activation
Basha Stankovich, Ph.D.
Bioservices Manager
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