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Shared Resources: Flow Cytometry and Cell Sorting

Policies and Procedures for DHVI Flow Facility Users

SOP Flow_010

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Standard Operating Procedure

SOP Number: SOP Flow_010 Effective Date: 8/1/2017

Version Issue Number: 4.1

Owner: Derek W. Cain, PhD

Author(s): Patrice M. McDermott, CCy Gregory D. Sempowski, PhD Derek W. Cain, PhD

SOP Flow_010 Title: Policies and Procedures for DHVI Flow Facility Users By signing the “Approved By” section below, the person attest that he/she has personally conducted a review of the document for completeness and accuracy and approves the contents of the SOP document.

Approved By: Derek W. Cain, PhD Signed: Date: 7/26/17 Title: Flow Facility Director, DHVI

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SOP Revision Document History:

Title & Version

Replaces

Effective Date

Description of Change

1.0 na 9/1/13 New SOP

1.0 1.0 2/4/14 Minor edit to update contact list in attachment

2.0 1.0 2/1/2015 Added Influx and Fortessa information as well as several edits throughout SOP

3.0 2.0 7/1/2016 Added Independent Sorter info

4.0 3.0 1/1/2017 Replaced FacMan content with CoreResearch content; updated links to new facility website

4.1 4.0 8/1/2017 Revised data management sections (section 10) to reflect retirement of Biotrue; updated cancellation policy for independent users (section 11.11); updated Attachment #1: Contact list

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Standard Operating Procedure

Policies and Procedures for DHVI Flow Facility Users

1.0 Purpose

The purpose of this SOP is to provide DHVI Flow Facility Users orientation to our resources, policies, and required procedures:

Section 2: History, Scope, and Application

Section 3: DHVI Flow Cytometry Facility Website

Section 4: User and project funding registration

Section 5: Hazard assessment forms

Section 6: Scheduling Cytometer Sessions

Section 7: Completion of experimental protocol sheets/templates

Section 8: Safe transportation of samples into Flow Facility laboratories

Section 9: Facility Entry/Exit Requirements and Proper PPE

Section 10: Data Management

Section 11: Overall policies

2.0 History, Scope and Application

The DHVI Research Flow Cytometry Facility serves the analytical and cell sorting needs of the Duke Human Vaccine Institute (DHVI) and researchers throughout the Duke Community. The Flow Cytometry Facility offers state-of-the-art cytometric support to investigators in basic, developmental, and clinical research.

The Flow Facility offers several cytometric services including BSL-1 through BSL-3 live cell sorting, phenotypic acquisition, DNA cell cycle analysis and intracellular marker analysis. Analytical and sorting capabilities up to 18 simultaneous parameters enable researchers to define subpopulations based on cell surface morphology as well as size and complexity.

The DHVI Flow Facility is partially supported by the NIH and conforms with guidelines established to ensure that the entire Duke Community has access to the Facility.

Investigators must be trained for independent use of the analytical instruments. The DHVI Flow Facility has offline analysis computer workstations available for data analysis with FlowJo software, an informational website (https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry), and a web-based scheduling and billing tool (CoreResearch).

This SOP applies to all DHVI Flow Facility End Users. Failure to comply with this SOP may result in loss of privileges to use DHVI Flow Facility services and instruments. This procedure does not provide instruction on running FACS Diva software (BD Biosciences) or the actual operation of the cytometers. Individual training with our facility staff is required for independently running our cytometers.

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3.0 DHVI Flow Cytometry Facility Website

3.1 URL: https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry

3.2 Instrument information, contact information, quick links to CoreResearch, user guides, and data archive links are present on the landing page and present on all pages in side panels.

3.3 “Get Started” Tab: Registration, Training request form, and Required Forms

3.4 “Instruments and Services” Tab: Instrument optical configurations, Training for independent use, and FlowJo software license seat request form

3.5 “Reservations and Rates” Tab: Link to CoreResearch, Link to facility to request rates

3.6 “Download Library” Tab: Links to End User SOP (this document), Instrument-specific protocol sheets, Instrument configurations, Quick user guides, Training materials, and Safety SOPs.

3.7 “Useful Info” Tab: Flow facility location, Policies and Procedures, Facility Acknowledgement, Resource links, Help & FAQs

4.0 User and project funding registration

4.1 The DHVI Flow Facility uses CoreResearch for billing. In order to use the DHVI flow facility, End Users must be linked to: 1) a Principal Investigator (PI), and 2) a project code (fund code in the CoreResearch@Duke system). Within CoreResearch, personnel with the following roles can make these linkages:

4.1.1 Principal Investigators

4.1.2 Principal Investigator-Delegates

4.1.3 Departmental Business Managers (person responsible for billing)

4.1.4 Flow Facility Staff cannot link End Users to PIs or projects.

4.1.5 For help with CoreResearch@Duke, visit https://sites.duke.edu/coreresearch/ or call Research Support Services at 684-2243, option 4.

5.0 Hazard Assessment Forms

The DHVI is fully committed to providing a safe work environment for the Flow Facility staff and its End Users. To assure the assignment of proper safety practices and procedures, it is necessary to collect detailed information about all potential samples being generated by a given research group prior to receipt.

5.1 An online Hazard Assessment Form must be completed and submitted for a given research group (i.e. laboratory or PI). These forms must be submitted online at least 48 hours prior to the inaugural job/session for a particular user or laboratory group. Typically, End Users or PIs accessing the facility for the first time complete one or more of these online forms to cover all potential sample types and situations for their research projects

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5.1.1 To fill out the online form, visit the “Get Started” page of the Flow Facility website and go to Step 4 (https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry/get-started)

5.1.2 Please complete one form for each sample species, pathogen, etc.

5.1.3 If assistance is needed, please contact the Facility Safety Officer (scott.alderman@duke.edu).

5.1.4 Forms can be submitted at any time to alert the Flow Facility staff to changes in sample risk/hazard.

5.1.5 Routine audits by DHVI Safety and Facility staff may result in a need for updated form submission.

5.2 Forms are reviewed electronically by the DHVI Safety Director. An appropriate containment level, and SOP, is assigned based on risk.

5.3 NOTE: More than one form may need to be submitted for a given PI to cover fixed/unfixed, infected/uninfected, and viral vectors/recombinant DNA.

5.4 Work will not proceed until safety approval is granted and appropriate containment level can be provided.

5.4.1 Projects assigned to BSL-3 and/or Select Agent requires comprehensive oversight by the Facility Director and the Duke Select Agent Principal Investigator. Please contact the Facility Director at least 4 weeks prior to initiation of any BSL-3 and/or Select Agent assigned work.

6.0 Scheduling Cytometer Sessions

ALL End Users are required to go through a one-time DHVI Flow Facility orientation before scheduling a session in CoreResearch@Duke. Please contact User Support (dhviflo@dm.duke.edu) to schedule an appointment.

6.1 Using an internet accessible computer launch a web browser (e.g. Internet Explorer, Google Chrome, or Safari but not Firefox).

6.2 URL: https://medschool.duke.edu/research/core-research-facilities

6.2.1 Click the blue button labeled CoreResearch@Duke.

6.2.2 Enter your NetID and Password, and press Enter.

6.2.3 If you do not remember your NetID, contact your departmental IT support.

6.2.4 If you are unable to log into CoreResearch, please contact Research Support Services at 684-2243, option 4.

6.2.5 Log in to CoreResearch as a Facility User.

6.3 Click on the “Reservation Calendar” tramstop in the Resource Reservation

tramline.

6.4 From the “Select Core” dropdown menu at top of screen, select “DHVI:

Flow Cytometry Facility”

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6.5 From the “Group” dropdown menu at top of screen, select the appropriate

reservation calendar:

6.5.1 Analyzers 24/7: to make a reservation for an analyzer (F01, L01, or

L02).

6.5.2 Sorters Mon-Fri: to make a reservation for a sorter (A01 or A02).

6.6 After the appropriate reservation calendar appears, navigate to the

desired date for a reservation.

6.7 To schedule a reservation, drag or double-click within the 24-hour

calendar for the appropriate day and instrument. A yellow block will extend

over the time period, then the Reservation Window will launch.

6.8 Complete the following fields in the Reservation Window:

6.8.1 Project ID: Click on the magnifying glass icon next to the field to

open the “Select Project” window, which shows the fund codes to

which you are linked. Within the “Select Project” window, press

“OK” in the Search Project ID/Project Title field to view all fund

codes. Click on the bullet of the appropriate Project, the click

“Select & Return” at the top of the screen. The Project ID field will

now be populated.

6.8.2 Reserved for Unique ID: This field should automatically be

populated with your Unique ID.

6.8.3 Reservation Start Date, Reservation End Date, Reservation Start

Time, Reservation End Time: Ensure that the correct date and time

are entered.

6.8.4 Activity: From the dropdown menu, select the activity associated

with your reservation. For a reservation on an analyzer, this

response will most likely be “Phenotyping (Independent).” For

sorting, this response will be “Sorting (Staff-Assisted).” Do not

select “Sorting (Independent)” unless you are appropriately

certified.

6.8.5 Membership: This field is located under the “Billing Info” tab!

Click on the magnifying glass icon to open the Select Membership

window, click on the “Duke” bullet, and then click “Select & Return.”

Click the Reservation tab to get back to the reservation info.

6.8.6 Experiment Information: For reservations on the F01, L01, or L02,

no information is required. For sorting reservations on the A01 or

A02, please provide the following information in the “Experiment

Information” box:

6.8.6.1 Sort Pressure (psi) and nozzle size (70, 85, or 100 μm)

6.8.6.1.1 70 psi/70 μm nozzle: lymphocytes for immediate

RNA, DNA, or protein isolation

6.8.6.1.2 55 psi/85 μm nozzle: for cell types that are 10-14

μm in diameter

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6.8.6.1.3 20 psi/100 μm nozzle: the gentlest sort condition;

recommended for large (14+ μm) cells OR

lymphocytes going into culture

6.8.6.2 Sort collection (e.g. 1-4 way, tubes, plates, slides etc.)

6.8.6.3 Species of cells (Human/NHP or not human/NHP)

6.8.6.4 Diva Template to be used, if available (e.g. “PBMC

template from 8/11/2016”)

6.9 Check all reservation information. At the top of the screen, click “Save.” If

any of the required fields listed in step 6.8 are empty, an error message

will occur – fill out the appropriate field, then click “Save” again.

6.10 If you have a Protocol Sheet ready, attach it to your reservation at this

time.

6.10.1 At the bottom of the Reservation, click on the tab for “Attachments.”

Click on the “Manage” button to open the Attachment Manager

window. Click on the “Add New” button.

6.10.2 In the newly created “Description” field, please type “Protocol

Sheet.” Click on the “Browse” button and then navigate to your

protocol sheet file. Select the protocol sheet file and click “Open.” In

the Attachment Manager window, the protocol sheet file name will

appear in the File field.

6.10.3 A dropdown menu will be available to the right of the File field.

Select “Shared Resources.”

6.10.4 Click “Save” at the top of the Attachment Manager window.

6.10.5 Click “Close” at the top of the Attachment Manager window.

6.10.6 The attached file should now be visible under the “Attachments” tab

of the Reservation window.

6.11 At the top of the Reservation window, click “Submit for Approval.” You will

receive an email from dhviflow@dm.duke.edu with a subject line

containing the Reservation ID and approval status. Open the attached

“.ics” file to create an appointment in your Outlook calendar. Note: If you

do not “Submit for Approval”, the reservation will be deleted after 10

minutes.

7.0 Completion of Experimental Protocol Sheets/Flow Templates

ALL End Users are required to fill out a current instrument-specific Experimental Protocol sheet for every experiment/session. For operator-assisted sessions, the protocol sheet must be emailed to dhviflo@dm.duke.edu 24 hours PRIOR to the scheduled experiment/session. For independent sessions, the protocol sheet must be completed and saved in the Experimental folder for data archiving.

7.1 All current instrument-specific forms or templates (Excel) are available from the Download Library on our website: https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry/downloads

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7.2 Forms are updated regularly so please go online every couple months and pull down a new template. Forms need to be filled out completely:

7.2.1 Required Information

7.2.1.1 Reservation ID: Enter the CoreResearch Reservation ID

7.2.1.2 Registered Lab PI/End User: Last name of the PI being invoiced/End User name or initials.

7.2.1.3 End User Telephone/Email: For contact purposes.

7.2.1.4 Human or Non-Human Primate?: To determine biosafety level for correct PPE to be used.

7.2.1.5 Fixed?: If no, your answer will result in “Contact Facility”. Call or email the facility (See Appendix 1) to discuss the appropriate plan of action.

7.2.1.6 Recombinant DNA/ Viral Vectors?: If yes, your answer will result in “Contact Facility”. Call or email the facility (See Appendix 1) to discuss the appropriate plan of action.

7.2.1.7 Select Agents?: If yes, your answer will result in “Contact Facility”. Call or email the facility (See Appendix 1) to discuss the appropriate plan of action.

7.2.1.8 Cell Type: PMBCs, Lymphocytes, etc.

7.2.1.9 Cell Line: Name of cell line, if applicable.

7.2.1.10 Sample Prep Date: Date the cells were prepared.

7.2.1.11 # of Events/Cells to Record: How many cells do you want us to capture for your data analysis? (Operator- assisted reservations only).

7.2.1.12 Record Gate: Which gate in the gating strategy do you want to use to collect recorded events? (Operator-assisted reservations only).

7.2.1.13 Sort Pressure: Default pressure (A01/A02) is 70 psi. Default sort pressure for the N01 varies. Any other pressure must be noted. Failure to provide custom pressure at least 24 hours prior to sort session will result in extra billed session time.

7.2.1.14 Sort Nozzle: Default nozzle (A01/A02) is 70 μm. Default nozzle for the N01 varies. Any other nozzle (85 or 100 μm) must be noted. Failure to provide custom nozzle requirement at least 24 hours prior to sort session will result in extra billed session time.

7.2.1.15 Sort Collection: Indicate how you will be sorting and what you will be sorting into. For example: 1, 2, 3, or 4-way sort (5 or 6-way for N01 only) into (1.5 mL, 5 ml, or 15 ml) tubes, 96-well plates, slides etc. Contact the Flow

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Facility if you need assistance in determining sort set up parameters.

7.2.1.16 Comments: Can be used to provide any additional information that you feel will assist the sorter Operator in running your session. Note: we cannot guarantee the same Operator for all sessions and therefore special request comments are essential for study continuity.

7.2.1.17 Detector: For each PMT detector, indicates the color of the laser being used as well as the mirror/filter configuration for that particular PMT (e.g. B-505LP_525_50 refers to a detector off the Blue laser with a 505 longpass filter followed by 525/50 bandpass filter). Note: Additional details regarding the optical configuration of each machine are shown on sheet 2 of the protocol workbook.

7.2.1.18 Expt. PMT Voltage: Allows you to record voltages used for PMTs from experiment to experiment after standardization of the first experiment.

7.2.1.19 Common Fluors: Examples of commonly used fluorochromes seen by each laser/detector configuration (see sheet 2 of the protocol workbook for additional fluorochromes that can be used).

7.2.1.20 Sample ID: Enter detailed description of what is in each tube (cell/bead/organism/antibody etc).

7.2.1.21 Tube #: Sequentially number all tubes 1 to X. Add additional lines if needed. Note: This is a requirement for Operator-assisted sessions.

7.2.1.22 Detector Channels: Enter the name of the actual fluorochrome used in each channel across the top of the grid (e.g.: FITC, PE, PE-Cy5 etc.).

7.2.1.23 Stains/Markers: please enter the name of the actual marker/stain being used in each fluorescent channel (e.g. CD3, CD4, Live/Dead etc.).

Note: We recommend that tube #1 on the protocol sheet be your negative control sample and your individual compensation tubes follow the order of the detectors being used on the protocol sheet from left to right to keep organized with the cytometer configuration setup.

7.2.1.23.1 Commercial compensation beads (kappa beads, Bangs beads etc.) can be a useful substitute for experimental cells. (Contact the Flow Facility for advice on panel development).

7.2.1.23.2 For more complex staining protocols/panels, we suggest also staining a series of tubes with all

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markers minus one (aka “FMO”). This allows for more robust and reliable compensation and analysis gating set up.

7.2.1.24 Sort Tubes: If a tube contains cells to be sorted, then please indicate the number of events to be collected.

7.2.1.25 Beads/Cells: This box can be used to keep track of FSC and SSC voltages used for setup beads vs. cells being used in the experiment.

7.2.1.26 Facility Operator Use: For Operator-assisted runs only.

7.2.1.27 Sort Setup (A01/A02/N01): Used to record the sort setup conditions for each sort, to compare from sort to sort.

7.2.2 For Operator-assisted sorts, email the completed Protocol Sheet to dhviflo@dm.duke.edu if it was not originally attached to the CoreResearch reservation.

7.2.2.1 If updating a protocol sheet, email the revised form to dhviflo@dm.duke.edu. Please enter “Updated protocol sheet” in the subject.

8.0 Safe Transportation of Samples into DHVI Flow Facility Laboratories

In accordance with Duke OESO and DHVI Safety protocols (see website), we require that all samples coming into DHVI Flow Facility laboratories be fully compliant with the following packaging/transport/labeling procedures:

8.1.1 At a minimum, all materials are to be double-packaged prior to transport. At least one layer of packaging must be leak-proof if the material is a liquid.

8.1.1.1 Example: placement of a leak-proof Vacutainer blood tube in a tube rack, and then placing the rack in closeable carrier (e.g. Igloo cooler).

8.1.2 For packages containing liquids, absorbent material must be included in sufficient quantity to absorb the entire contents of the primary container should a spill occur.

8.1.3 The outer sides of the carrier must be sprayed (prior to transport) with a disinfectant that is effective against the potentially infectious material(s) used in the laboratory. Hard plastic exteriors are best for cleanability. Once this step is complete, the outer surfaces of the carrier are considered safe for handling with bare hands. Lab coats and gloves should not be worn when transporting carriers outside the laboratory.

8.1.4 A biohazard warning label shall be applied when applicable to the carrier to communicate the potential hazard of its contents. Any potentially infectious material (e.g. human blood, unfixed human tissue, bacterial culture) would require the biohazard label (see example).

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9.0 Facility Entry/Exit Requirements and Proper PPE

9.1 When entering a cytometer room, closed toed shoes MUST be worn.

9.2 When using the cytometer, proper PPE must be worn by all Users sitting at the cytometer. This includes:

9.2.1 A white lab coat and gloves under Green status.

9.2.2 A blue lab coat, double gloves and a face shield under Red status.

9.2.3 Note: Before entering the cytometer room, PPE for Red status must be put on in the anteroom and the Biomedical Research Laboratory Status sign turned from Green to Red status.

9.3 When exiting the cytometer room under Green status, gloves must be removed and disposed of in the Biohazard container, lab coat removed, the cooler sprayed with 70% ethanol and hands washed thoroughly with soap and warm water.

9.4 When exiting the cytometer room under Red status, the outer gloves must be removed and disposed of in the Biohazard container and the cooler sprayed with 70% Ethanol before entering the anteroom. In the anteroom, lab coat is removed, gloves removed and disposed of in the Biohazard container, the Biomedical Research Laboratory Status sign turned from Red to Green status, and hands washed thoroughly with soap and warm water.

10.0 Data Management

10.1 Data Transfer: Data acquired during operator-assisted and independent user runs will be transferred to users via Duke Box.

10.1.1 Operator-assisted sessions: The operator will email the user a link to experiment-specific data (FCS files, sort reports, etc.) stored on the DHVI flow facility’s Box account. Users must download this folder from the link within 48 hours.

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10.1.2 Independent user sessions: Users will export their Experiments from Diva then upload the Experiment folder to their individual Duke Box account. Storage on Box accounts is limited to 50 GB – users are responsible for downloading Experiment data for analysis and storage according to lab-specific data management policies.

10.2 Data Archive: We regularly and automatically archive all flow experiments, allowing for the retrieval of FCS files, protocol sheets, sort reports, etc. Independent Users are responsible for exporting their Experiments to the correct workstation folder for data archiving.

10.3 Data Analysis: We use and recommend FlowJo for off-line flow data analysis. The DHVI Flow Facility manages a Duke site license for FlowJo. A seat on this license is purchased annually (July 1-June 30) and can be requested via our web page (https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry/instruments-services/duke-flowjo-site-license).

11.0 Overall Facility Policies

11.1 End Users caught violating ANY safety protocol will have immediate suspension of Facility use. The End User must meet with the Facility Director and the DHVI Safety Director to determine future use.

11.2 End Users that have three documented violations of our policies will have Facility use suspended and must meet with the Facility Director to determine future use. If needed, the User’s PI will be included in these discussions.

11.3 It is critical that the authorized End User select the correct Project for the experiment being run to maintain cost compliance with federal grants and a safe environment. If you are unsure - ask your PI!

11.3.1 If the project code needed is not listed when making a reservation in CoreResearch, then the End User needs to be linked to that project. Please contact your PI and/or financial administrator.

11.4 Sessions cannot be scheduled under another End User’s name. If the End User is not registered in CoreResearch, then please contact your PI and/or financial administrator.

11.5 Analytical cytometers (L01, L02, F01) can only be operated by trained End Users. Trained Independent End Users cannot allow their untrained colleagues to operate a cytometer independently. To become a trained independent End User, please go to the DHVI Flow Facility website and get on the training wait list: https://shared-resources.dhvi.duke.edu/dhvi-core-facilities/dhvi-flow-cytometry/get-started.

11.6 Sorting cytometers (A01, A02 and N01) are operated by a DHVI Flow Facility Operator or an approved Independent Operator. Sort samples take priority over phenotyping samples.

11.7 End Users are responsible for scheduling their cytometer sessions in CoreResearch. When scheduling a sort session, please account for an

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additional 30 minute setup time for the default pressure/nozzle setup (see 7.2. above) or an additional 45 minutes for a custom setup.

11.8 Restricted Sorting cytometers (A01-Restricted Access, A02- Restricted Access calendars) are booked by those who have been APPROVED by the DHVI Flow Facility Director for independent sorter operation. These Independent Operators are responsible for scheduling their sessions in CoreResearch on these cytometers.

11.9 The BD FACS Aria (A01) is designed for high-containment sorts as well as standard sorts. It is housed in the NIAID-Regional Biocontainment Building at Duke called GHRB. To access the BD FACS Aria (A01) in GHRB 1071, you will need a valid DUKE ID or government issued ID to clear Security. Users will be screened by Duke Security before entering the building (similar to airport security).

11.10 To access the BD FACS Aria (A02) in MSRBII 4036 or the BD Influx (N01) in MSRBII 4038, you will need to phone the Flow Facility 919-684-4130 so someone can let you into the 4th floor hallway (there is a phone next to the elevator that you can use).

11.11 Canceling sessions:

11.11.1 Operator-Assisted sessions: Up to 24 hours before a scheduled session, you can log into CoreResearch and cancel your session with no penalty.

11.11.1.1 If you are running late or need to change/cancel your scheduled time slot within 24 hours of the start time, then contact the Flow Facility (919-684-4130) so that we can act accordingly. If there is no answer at that number, email the DHVI Flow Cytometry mailbox at dhviflo@dm.duke.edu. Additional contact numbers are in Attachment 1.

11.11.1.2 If you do not show or if you cancel inside the 24-hour window, you will be invoiced for that scheduled time unless we can find another End User to fill the time slot. Contact the Facility Director if you have extenuating circumstances.

11.11.2 Independent End User sessions: Independent Users can cancel their sessions at any time.

11.11.2.1 Note: If you are the last user of an instrument that day, and you cancel after all earlier sessions have concluded – you are still responsible for shutting the instrument down.

11.12 If you require a Facility Operator to assist with your “Independent” session in excess of 15 minutes, or require routine troubleshooting, you will be invoiced for the Operator’s time.

11.13 Independent End Users: All sessions MUST be scheduled in CoreResearch before being run on the cytometer.

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11.14 Logging instrument time for Independent Users: Sessions are invoiced by CoreResearch from the earliest Actual Scheduled Start time to the Latest Scheduled End time. 11.14.1 If you begin and end your data acquisition within the original

reservation window, you will not need to do anything - CoreResearch will automatically bill the appropriate fund code for the scheduled duration of the reservation.

11.14.2 If you begin your session before the reservation start time and/or your session extends past your reservation end time, you will need to modify your actual usage. Here are the steps to do this: 11.14.2.1 Upon completion of your data acquisition, log into

CoreResearch at the cytometer workstation (See Section 6.2).

11.14.2.2 Navigate to your reservation on the Reservation Calendar, and double-click on it.

11.14.2.3 On the menu at the top of the screen, click “Update Actual Usage.”

11.14.2.4 Change the “Actual Start Time” and/or “Actual End Time” to reflect your early start or late stop. Note: you will not be allowed to make the “Actual Start Time” later than the original reservation start time, nor the “Actual End Time” earlier than the original reservation end time.

11.15 There is a 30-minute minimum schedule time for a session and a session will be paused by Facility staff during instrument malfunction or with approval of the Facility Director.

11.16 It is the responsibility of the Independent End User to export their Experiments files from FACS Diva to the appropriate workstation folder for data archiving. The Facility will maintain an archive of all Diva experiments but we cannot guarantee immediate retrieval.

11.17 FlowJo site license seats are available and are invoiced annually on July 1st. Initial purchases are prorated for the number of remaining months on the annual seat. NO REFUNDS for seat cancellation.

11.18 The DHVI Research Flow Facility is supported by a variety of shared instrumentation grants, center grants, and program awards. Acknowledgement of our facility and applicable funding in publications demonstrates our utility to Duke and funding sponsors. A list of example acknowledgment text can be found in Attachment 2.

11.18.1 Please include all funding affiliations that apply. If you have a question about the appropriate grant to acknowledge please contact Flow Facility Director.

11.19 The DHVI Research Flow Facility reserves the right to cancel or reschedule booked sessions based on a priority list drafted by the Facility Advisory Board.

12.0 Attachments

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12.1 #1: DHVI Flow Facility Contact List

12.2 #2: Facility Acknowledgement

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Attachment #1: DHVI Flow Facility Contact List

General Flow Facility Support 919-684-4130

CoreResearch Support Services 919-684-2243 option 4

MSRB2 Help 919-452-2709

Labs/Instruments

A01 GHRB, 1071 919-684-8813

A02 MSRB2, 4036 919-613-5920

N01 MSRB2, 4038 919-681-0629

F01 MSRB2, 2045 Bay 6 919-684-4130

L01 MSRB2, 2045 Bay 5 919-684-4130

L02 MSRB2, 2045 Bay 6 919-684-4130

Role Faculty/Staff Office Phone

Facility Director Derek Cain, PhD 919-681-8193

Flow Engineer Patrice McDermott, CCy 919-684-4130

Flow Engineer Steven Slater, CCy 919-684-4130

Billing/Finance Darlene McCain 919-681-1783

Scientific Advisor, CHAVI-ID Tony Moody, MD 919-668-2551

Scientific Director, DHVI Shared Resources Gregory Sempowski, PhD 919-684-4386

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Attachment #2: Facility Acknowledgement

General Facility Acknowledgement:

“Flow cytometry was performed in the Duke Human Vaccine Institute Research Flow Cytometry Facility under the direction of Dr. Derek W. Cain (Durham, NC).”

For Work performed in the GHRB (NIAID Regional Biocontainment Laboratory at Duke; A01) please add the following:

“Research was performed in the Regional Biocontainment Laboratory at Duke which received partial support for construction from the National Institutes of Health, National Institute of Allergy and Infectious Diseases (UC6-AI058607).”