measurement of CTL activity
In vivo priming of mice with antigen
- Inject mice subcutaneously with appropriate dose giving 80%
tumour incidence within 2 weeks of challenge (5x10^6 for MOPC-21).
- Monitor tumour progression 3 times weekly.
In vitro expansion of precursor CTL
- Remove spleen and make cell suspension by mechanical
disruption with glass slides.
- Lyse* red blood cells by adding 1ml of RBC lysis buffer
(Sigma) at RT for 1min.
- Wash and resuspend spleen cells at 8x10 6/ml.
- Irradiate 5x10^6/ml stimulator cells (12000 rads for MOPC-21)
to block cellular division.
- Wash 3 times and resuspend stimulator cells at 4x10^6/ml.
- 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.
- Add 1ng/ml recombinant murine IL-2 (BD) per well.
- Incubate in a humidified chamber at 37°C with 5% CO 2 for 5
*Not necessary if LN used as responder.
** Responder: stimulator= 4x10^6: 2x10^6 for LN.
In vitro detection of CTL: 51Cr Release Assay
- 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.
- 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
- Wash labelled target cells 3 times and resuspend at
1x10^5/ml. Proceed to step 7 immediately.
- 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
- 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.
- To determine minimal and maximal release, add 100uL of media
or 100uL of 1% Triton X-100 to each well in triplicate.
- 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.
- Seal plate and centrifuge at 1500 rpm at 4 ° C for 5min to
- 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.
- To calculate the mean percentage specific lysis:
= [(Sample cpm- minimal cpm)/Maximal cpm - minimal cpm)]x 100%
*Use filter tips and lead shielding when handling