Induction and measurement of CTL activity

Contents:

 

 

 

In vivo priming of mice with antigen

  1. Inject mice subcutaneously with appropriate dose giving 80% tumour incidence within 2 weeks of challenge (5x10^6 for MOPC-21).
  2. Monitor tumour progression 3 times weekly.

 

In vitro expansion of precursor CTL

  1. Remove spleen and make cell suspension by mechanical disruption with glass slides.
  2. Lyse* red blood cells by adding 1ml of RBC lysis buffer (Sigma) at RT for 1min.
  3. Wash and resuspend spleen cells at 8x10 6/ml.
  4. Irradiate 5x10^6/ml stimulator cells (12000 rads for MOPC-21) to block cellular division.
  5. Wash 3 times and resuspend stimulator cells at 4x10^6/ml.
  6. In a 24-well flat-bottom plate, culture 8x10^6** spleen cells per well in the presence of absence of 4x10^6 stimulator cells in a total volume of 2ml.
  7. Add 1ng/ml recombinant murine IL-2 (BD) per well.
  8. Incubate in a humidified chamber at 37°C with 5% CO 2 for 5 days.

*Not necessary if LN used as responder.

** Responder: stimulator= 4x10^6: 2x10^6 for LN.

 

In vitro detection of CTL: 51Cr Release Assay

  1. Resuspend 1x10^6 target cells in 150uL and label with 150uCi (150uL of 1mCi/ml) of sodium chromate ( 51Cr*) (Perkin Elmer), ie. final concentration at 3.33x10^6/ml.
  2. Incubate with cap loosened in a humidified chamber at 37 ° C with 5% CO 2 for 1hr to allow uptake of 51Cr. Gently resuspend cells once during incubation to increase uptake. Proceed to step 4 during incubation.
  3. Wash labelled target cells 3 times and resuspend at 1x10^5/ml. Proceed to step 7 immediately.
  4. Perform cell count with ethidium bromide/ acridine orange and resuspend 1.35x10^6 spleen cells in 450uL. In a 96-well round-bottom plate, transfer 150uL of spleen cell suspension to each well in triplicate for the first effector: target ratio. Add 100uL of media to subsequent wells.
  5. Using a multichannel pipette, serially dilute 3-fold by transferring 50uL across 6 wells in triplicate, discarding the final 50uL. Hence E:T ratios should be 30:1, 10:1, 3:1 1:1, 0:3:1, 0.1:1.
  6. To determine minimal and maximal release, add 100uL of media or 100uL of 1% Triton X-100 to each well in triplicate.
  7. Add 100uL of 51Cr-labelled target cells to each well and incubate in a humidified chamber at 37 ° C with 5% CO 2 for 4hr.
  8. Seal plate and centrifuge at 1500 rpm at 4 ° C for 5min to pellet cells.
  9. Using a multichannel pipette and proceeding from replicates of low-level 51Cr to high-level, aspirate 100uL of supernatant without disturbing the pellet, into Titretube micro tubes (Bio-Rad) for counting on a Packard-Cobra autogamma counter. If using scintillant-coated LumaPlate, aspirate 25uL of supernatant into each well for counting on a TopCount microplate scintillation counter.
  10. To calculate the mean percentage specific lysis:

%Specific lysis = [(Sample cpm- minimal cpm)/Maximal cpm - minimal cpm)]x 100%

*Use filter tips and lead shielding when handling 51Cr.