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SOP Nr AI/CTL/05 Revision Nr


Peptide Stimulated 51 Cr Release Assay


DATE IMPLEMENTED 01 December 2003






APPROVAL OF STANDARD OPERATING PROCEDURE Requires the signatures of the following persons : Principle investigator or designate Signature : ………………………………… Date : …………………………………

Dr Clive Gray


Signature : ……………………………….. Date : ………………………………..

Greg Khoury


DISTRIBUTION LIST Designation Signature


Acute Infection Project

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1. Purpose:
This is descriptive procedure detailing the set-up and execution of the 51Cr release to measure Cytotoxic T Lymphocyte (CTL) activity following a 14 day in vitro stimulation of peripheral blood mononuclear cells.

2. Scope:
This protocol is applicable to all staff in the Immunology Lab performing CTL assays

3. Principles
The CTLs Assay is used to detect the cytolytic activity of Ag-specific lymphocytes. The classical assay for CTL activity is the chromium release assay. CTL’s function by destroying cells those express foreign antigens (Ag) such as virally infected cells: typically mediated by CD8+ T-lymphocytes. The basic requirements for CTL detection involve both a source of HIV-1 specific CTL effectors and an appropriate target cell expressing the HIV-1 encoded viral antigen(s) of interest. Target cells expressing foreign Ag on their surface are labelled with radioactive isotope of chromium (51Cr)-Hot targets. In this Assay, Target cells are autologous EBV (Epstein Barr Virus) Transformed B Lymphoblastoid cell line (BLCL) that expresses appropriate HIV Ags following in vitro infection with recombinant Vaccinia viruses. EBV is able to enter B-lymphocytes because of its receptors and following the expression of its oncogenes can transform normal B cells and immortalize them. T-lymphocytes from EBV seropositive individuals suppress B-cell immortalization by EBV. Thus, immortalization of human B-Lymphocytes by EBV occurs with greater frequency if the EBV- specific T-lymphocytes are removed from culture or are functionally inactivated with a specific T-lymphocyte immunosuppressive compound Cyclosporin A. Autologus B cells, used as targets are thawed two weeks before the assay, to allow growth. Patient effector cells (Effectors) are then mixed with target cells and incubated several hours. Lysis of Ag-expressing target cells releases 51Cr into the medium. This is detected on the Topcount analyser. HIV specific Lysis is calculated by comparing lysis of target cells expressing HIV or control Ags in the presence or absence of patient effectors cells. Effectors are peripheral blood lymphocytes (PBMC), obtained by ficoll separation, that have been stimulated for 14 days, in the in vitro stimulation (IVS) cultures. The goal of the IVS is to increase in vitro the number of CTL elicited by the in vivo immunisation, to a level detectable in the standard 51Cr release Assay. In IVS a portion of PBL is used as responder cell population. These cells should specifically proliferate in response to an antigenic stimulus.

Acute Infection Project

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Two cytokines are important in the amplification of the CTL response during the IVS (IL-2 & IL-7). Their effect is dependent on the time of addition to the culture. Human recombinant IL-7 is usually added the first day to help in increasing the frequencies of CTL precursors and it increases Ag processing and presentation by professional Ag presenting cells. Human IL-2 is usually added on day 7 to support the cell growth in the second week of IVS.

4. Safety Procedures
4.1. Standard safety-operating procedures are to be followed at all times. 4.2. Treat all materials as infectious, 4.3. All procedures (unless otherwise stated) are to be performed in a Biohazard Class II safety Cabinet. 4.4. White coats, double gloves, Blue surgical gowns, safety glasses and overshoes must be worn at all times. 4.5. Always wear radioactive protective clothing when working with radioactive materials. 4.6. Wear Finger rings and carry a dosimeter in your pocket. Note: It is important to have finger ring and the dosimeter on you at all times while working with radioactive materials. Never ever leave them anywhere in the radioactive Laboratory.

5. Reagents and Materials:
5.1. RPMI 1640 (Gibco) 5.2. FCS (Gibco HI Fetal Calf Serum -heat inactivated at 56˚C for 45 minutes). 5.3. PBS (Gibco Dulbeco’s Phosphate Buffered Saline) 5.4. Gentamicin (Gibco) 5.5. 51Cr 5.6. Liquid Waste container 5.7. Radioactive Waste container 5.8. Virocide solution 5.9. 15ml conical centrifuge Tubes 5.10. 50ml conical centrifuge Tubes 5.11. 25ml tissue culture flasks with filter caps (T25) 5.12. Plastic Counting Chambers Acute Infection Project

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SOP Nr AI/CTL/05 Revision Nr 5.13. Trypan Blue solution 5.14. Microscope 5.15. Centrifuge 5.16. Pipette Aids 5.17. Pipettes: 2ml, 5ml, 10ml, 25ml 5.18. Pipette tips: 10ul; 20ul; 100ul; 200ul; 1000ul 5.19. Dynal capture magnet 5.20. Dynal magnetic beads:anti-IgG, anti-CD4, anti-CD8 5.21. Poplypropylene tubes 5.22. Lead Apron 5.23. Shield 5.24. Finger ring 5.25. Dosimeter


6. Media Preparation
6.1. R 1 1% RPMI FCS 6.1.1. 500 ml RPMI 6.1.2. 5.5 ml HI FCS 6.1.3. 500ul Gentamicin 6.2. R 10 10% RPMI FCS 6.2.1. 500ml RPMI 6.2.2. 55 ml HI FCS 6.2.3. 500ul Gentamicin 6.3. R 20 20% RPMI FCS 6.3.1. 500ml RPMI 6.3.2. 110ml HI FCS 6.3.3. 500ul Gentamicin

7. 51 Chromium Activity Calculations
To calculate the volume required to label the HOT targets you need to have: 7.1. Activation date, 7.2. Concentration on activation day, Acute Infection Project

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SOP Nr AI/CTL/05 Revision Nr 7.3. Days from reference and factor 7.3.1. Concentration of 51 Cr required for the assay [ ] on assay day =Factor x [ ] on activation day For this Assay, 100 uCi/sample is required. Volume required = [ ] required per sample [ ] on Assay day


8. Method
8.1. Preparation of B Cells 8.1.1. B Cells must be resuscitated at least 2 weeks before the CTL Assay. Thaw cells as per thawing SOP. 8.1.2. Resuspend cells in 5ml fresh R20 and transfer the cells to a T25 flask. Allow the cells to rest overnight at 37˚C and feed the next day with R20 (50% media exchange). 8.1.3. Maintain the cells in the growing stage by feeding every three to five days and allow the culture to expand so that there will be sufficient cells for the assay 8.1.4. 8.1.5. Feed the cells for the last time 24 hours before the CTL assay The day before the assay, mix cells well and measure the volume of suspension with a pipette. 8.1.6. 8.1.7. 8.1.8. 8.1.9. Perform a viable cell count on the BLCL using trypan blue. Count both viable and nonviable cells and record the cell count. A minimum of 1 x10e6 B Cells are required as HOT targets Pellet the cells at by centrifugation at 1200 rpm for 10 minutes and resuspend to 1ml. Decant the supernatants and resuspend the Hot Targets in 1ml of warm R20 to a final concentration of 1 x 10e6 cells/ ml 8.1.10. Add 100uCi 51Cr per vial and pulse the peptides with 10ug/ml peptide 8.1.11. Incubate with loosened caps for 90 minutes in a 37˚C, 5% CO2 incubator. 8.2. Preparation of the effectors 8.2.1. 8.2.2. 8.2.3. 8.2.4. 8.2.5. Remove the 14 day IVS from the incubator Re-suspend the effectors with a pipette to obtain a single cell suspension. Measure the volume of the culture and record it. Perform a viability count the cell count. T cell depletions are to be performed on the cells to remove CD4+ Cells and CD8+ Cells. In IgG Isotype control is included. Prepare two sets of 15 ml polypropylene tubes: one for the depletions and one for washing the cells after the depletion. Three tubes are required in each set: labelled as follows: PID, IgG , PID, CD4 PID, CD8 Acute Infection Project

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SOP Nr AI/CTL/05 Revision Nr 8.2.6.


Write on the first set of tubes, the volume of the IVS cell suspension that must be removed and the volume of beads that must be added to the tube


Write on the second set of tubes the volume that the suspension must be resuspended to for the first E:T ratio.


The minimum number of cells required per depletion is 1.5x106, thus the 3 depletions will require a minimum of 4.5x106 cell.


Prepare and label two sets of three 14ml tubes to wash the magnetic beads in.

8.2.10. The first set to wash beads before adding to the cells 8.2.11. The second set to collect beads from cells after incubation. 8.2.12. The total number of cells require to fulfil this assay is 1.5x106. 8.2.13. For 50:1 effector: target ratio in triplicate, you need 750 000 cells~250 000 cells/well. 8.2.14. Therefore 25:1 effector: target will require half of 50:1 which is 375 000 cells~125 000 cells/well. 8.2.15. 10 beads~1 cell, then for depletion, 15x106 beads will be required. 8.2.16. To calculate the number of beads required for the assay, Vol of Beads To Remove = Beads required x the number of samples you have Beads [ ]/ml 8.2.17. Put beads in pre-prepared magnetic beads tubes with 8ml cold 1% RPMI and place them in the capture magnet. 8.2.18. Remove the supernatant with a pipette and remove tubes from the capture magnet. 8.2.19. Repeat the first step three times and after the third wash re-suspend the beads with cold 1% RPMI. 8.2.20. Aliquot the appropriate number of cells in each of the three 15ml conical tubes (labelled as anti-IgG, anti-CD4 and anti-CD8) , and balance the tubes by adding cold 1% RPMI. 8.2.21. Spin at 1200rpm for 10 minutes and decant the supernatants in the non- infectious waste container. 8.2.22. Add 1ml of the beads suspension such that you have 10 beads per cell. 8.2.23. Mix gently: put the tubes on a mixer and incubate for 45 minutes at 4 ˚C. 8.2.24. At the end of the incubation, add 4ml of 1% RPMI and place the tubes in the capture magnet. 8.2.25. Let the tubes stand for at least 2 minutes in the capture magnet, then, with the tubes still in the capture magnet, remove the supernatant and transfer to the appropriate 15ml conical washing tubes. 8.2.26. Make a 1:2 dilution of anti-IgG –IVS and trypan blue and do call count. 8.2.27. Record the cell count. 8.2.28. Wash effectors for 10 minutes at 1000 Rpm 8.2.29. Decant the supernatant in the non-infectious liquid waste container. 8.2.30. Re-suspend them at 2.5x106/ml with 10% RPMI. 8.2.31. Cells are ready to be plated in triplicates at 50ul/well. Acute Infection Project

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SOP Nr AI/CTL/05 Revision Nr 8.3. Wash hot Targets 8.3.1. 8.3.2. 8.3.3. 8.3.4. Add 9ml of PBS to the tube and wash once at 1200rpm for 10 minutes. Decant the supernatant in the radioactive liquid waste container.


Gently re-suspend the cells with a tip, add 10ml PBS and wash twice, discarding supernatants in the radioactive waste container. After the third wash re-suspend the cells in 5ml of 10% RPMI to obtain a single cell suspension.


Do cell count.

8.4. Adjust the cell concentration to 5x104 cells/ml with 10%RPMI 8.4.1. Cells are ready to be plated at 50ul/well in order to have 2500 targets per well.

8.5. Plating cells 8.5.1. 8.5.2. 8.5.3. 8.5.4. 8.5.5. Mark the plate and start plating in this order: Pipette 50ul of 10%RPMI in the wells for the spontaneous release. Pipette 50ul of 1% triton solution in the wells for the maximum release. Pipette 50ul of effector cells of the highest E:T ratio for each bead concentration. Double the left over volume of the effector cell suspension and plate the second E:T ratio accordingly. 8.5.6. Plate 50ul of the HOT target cells in every well starting from Spontaneous release and ending with Maximum release. 8.5.7. 8.5.8. 8.5.9. Incubate the plate for 4 hours at 37˚C Label the plate with harvest time. At the end of incubation, harvest 25ul of the medium and transfer to Lumaplate for counting.

8.5.10. Leave the plate to dry overnight and read the next day.

Acute Infection Project

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