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Cytotoxic T-Lymphocyte Assay

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > Assays > Other Assays


Branch: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
24.06.2010 - John Stewart   


A CTL Assay will indicate the presence and cytotoxic activity of T cells to a specific antigen. The following assay is based upon Chromium release following MHC CI presentation (H-2b???) of the SIINFEKL peptide to cells primed with the OVA antigen. Other labelling, other MHC CI, other peptide and other antigen is of course possible, providing it is thought completely through.

Contents

[edit] Overview

Generally on day 0, the animal is primed with antigen of interest, and then T cells derived for bulk culture (see below) from the spleen on day 7. Longer and more complex priming strategies are of course necessary for some antigens, and the spleen is not always necessary / viable.

On day 11, EL4 target cells are loaded with 54Cr and cultured overnight with T cells from bulk culture. The following day, the amount of chromium release is assayed.

The following assay assumes the use of the C57 Black 6 (C57BL/6) mouse model, but should work in any model or models where the MHC CI matches.

[edit] Bulk Culture for CTL Assay

[edit] Preparation of Effector Cells

  1. Remove spleens from treated animals, into ~10mls RPMI + 2.5% FCS; and take at least 1 control spleen per 2 treatment spleens.
  2. Make a single cell suspension by gently pressing spleen cells through a sterile sieve into RCRB (8.24g/l NH4Cl, 37mg/ml EDTA, 1g/l NaHCO3); leave for 5’ at room temperature before putting on ice.
  3. Spin for 5’ at 1.5K at 400C; and resuspend in 10 mls cold RPMI + 10% FCS (keep on ice).

[edit] Preparation of Stimulator Cells

  1. Remove 1 spleen per 2 treatments from C57BL/6 mice and placed in a single 10ml tube with RPMI +10% FCS.
  2. Make a single cell suspension by gently pressing spleen cells through a sterile sieve into RCRB (8.24g/l NH4Cl, 37mg/ml EDTA, 1g/l NaHCO3); leave for 5’ at room temperature before putting on ice.
  3. Spin for 5’ at 1.5K at 400C; and resuspend in 2 mls RPMI + 2.5% FCS.
  4. Irradiate cells at 1500 rads.
  5. Incubate irradiated cells with 2ul filtered peptide [0.1mg/ml] per spleen (0.2uM).
  6. Wash 3 times with 10mls serum free media; and finally resus in 10mls RPMI + 10% FCS.

[edit] Bulk Culture

  1. Suspend 3 x 107 Effector cells with 3 x 107 stimulator cells in a total of 30 mls RPMI + 10% FCS.
  2. Incubate flask for 6 days at 370C with 5% CO2.

[edit] 51Chromium Release Assay

[edit] Load Target Cells

The following assay should be performed in a radiation lab following all safety rules, regulations and requirements. Consult your radiation / safety officer before carrying out this experiment. WEAR GLOVES & GLASSES; USE FUME HOOD; DISPOSE OF WASTE APPROPRIATELY; and OBSERVE ALL SAFETY REQUIREMENTS.

  1. Prepare EL4 cells to a concentration of 107 cells /ml in RPMI +10.
  2. Remove 2 X 200ul aliquots to two tubes, each containing 200uCi 51Cr.
  3. Add 8ul target peptide (from conc. [.1ug/ul]) to one tube only (Target Cells). This concentration of peptide may require higher concentrations, depending on binding efficiency. The other tube is for Non-Specific Lysis Controls (Non-peptide loaded Irradiated Cells).
  4. Incubate with lids on tight, in lead pegs 1.5 hrs at 370C; flick tubes to resuspend cells every 30’.
  5. Meanwhile: Prepare Responder Cells (see below).
  6. Wash three times with RPMI + FCS 5% and dispose of waste properly.
  7. Wash irradiated Target Cells once more; resus in 20 mls RPMI + 10% FCS (to final conc. [5 X 105cells/ml].

[edit] Prepare Responder Cells

  1. Centrifuge Bulk Cultured cells for 5' at 2K; resuspend at 107cells/ml in RPMI + 10% FCS.

[edit] Prepare Assay Plate and Controls

Use a 96 well U bottom plate, with letters down the side (rows) and numbers across the top (columns). A good system / layout for samples is to load empty media into all rows except the top row (A), so that you can titrate Responder Cells down the plate to the second bottom row (G). The bottom row (H)  used for Non-Specific Lysis Controls (see below). The final two columns of one plate can be assigned for the (very important) controls (see below).

  1. Aliquot 0.1ml RPMI + 10% FCS into rows B through to F or G.
  2. 4 x Total 51Cr Controls:  100ul (RPMI + 10% FCS) + 100ul Irradiated Target Cells; A11 to D11.
  3. 4 x Spontaneous Lysis Controls: 100ul (RPMI + 10% FCS) + 100ul Irradiated Target Cells; E11 to H11.
  4. 4 x Max Lysis Controls: 10ul TritonX-100 + 90ul (RPMI + 10% FCS) + 100ul Irrad Target Cells; A12 to D12.
  5. 2 x Non-Specific Lysis Controls: 100ul Responders + 100 ul Non-peptide loaded Irradiated Cells; duplicates for each treatment of responders in bottom row (H).

[edit] Chromium Release

  1. Transfer 200ul responder cells to top row and 100ul  bottom rows (A and H) in duplicate (this will ultimately leave 106 cells in these wells) .
  2. Titrate 100ul from top row (A) down plate to second bottom row (G) (ie. serial dilutions). Higher dilution factors may be required, such as 1:3, 1:5 or even 1:10, if significant CTL activity is expected.
  3. Incubate 4 hours at 370C.
  4. Re-suspend the cells from the Total 51CR Controls and remove 100ul aliquots to separate measurement tubes; and centrifuge remaining plate contents for 5’ at 2K.
  5. Remove 100ul S/N to measurement tubes; and incubate o/n at 650C (drying out the contents).
  6. Place measurement tubes into racks and into machine (eg, Hitachi), and follow operating instructions.

[edit] Results

The Chromium Release Assay can be assumed to have been effectively performed if: there is relatively little Cr release from Spontaneous Lysis Controls; and the Max Lysis Controls is almost as high as the Total 51Cr Controls.

Specific cytotoxic activity can be said to be demonstrated if Chromium release decreases down the titrated columns in the antigen stimulated animals, but not down the control animals, AND there is a similar level of Chromium release in the Non-Specific Lysis Controls as n Spontaneous Lysis Controls.

The typical way to present results is as the "percent of specific lysis" calculated via the following formula:

% Specific lysis = [(Sample cpm - Spontaneous Lysis Control cpm)/Maximum Lysis Control cpm - Spontaneous Lysis Control cpm)] x 100%

This can then be plotted as a line graph with % specific lysis on the y-axis, and dilution on the x-axis, and should show untreated controls as well as various treatments.

[edit] Acknowledgements

The basis of this assay was kindly explained by Justine Mintern (WEHI) to Christopher Dyer over many patient months during their respective PhDs.





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