Killing Assay

2015-01-13

azim58 - Killing Assay

Killing Assay

E:T ratios (3 replicates)

o Note that the T cells could come from a 5 day induction or
straight from the spleen
o 100 (100/1=10^6/y -> y = 10^6/100 = 10^4) (10^6 effectors to 10^4
targets)
o 30 (10^6 effectors to 3.3*10^4 targets)
o 10 (10^6 effectors to 1*10^5 targets)
o Note that unusually active CTL can result in detectable lysis at
effector:target ratios <1:1. Concentrations >2*10^6 cells/well
commonly suppress activity.

Controls

o Target cells not pulsed with peptide mixed with effector cells
o Target cells pulsed with peptide mixed with effector cells that
have not been "induced
o Target cells alone
o effector cells alone
o spleen cells and peptide alone
o other possible controls:
- Non-enriched T cells (naive and immunized)
- Plain splenocytes with no 5 day induction (naive and
immunized)

Determine cells needed

o Approximate total number of effectors: 1*10^6 * 7 * 3 = 2.1*10^7
cells
o Approximate total number of targets: 1*10^4 * 4 * 3 + 3.3*10^4*3
+ 1*10^5 * 3 + 3.3*10^5 * 3 = 1.5*10^6 cells
o Note that adherent cells should not be detached with trypsin
since this could remove cell surface markers such as MHC molecules
with peptide. Perhaps hyQtase can be used to detach the target cells
instead of trypsin.
o If cells are forming clumps, they may need to be broken by
filtering them through a 40 um nylon mesh.

Pulse target cells with peptide. This can be done by adding peptide

to a final concentration of 10 ug/mL and incubating at 1-4 hr for 37 C
o Ex calculation: 1000 ug/mL * y / (5 mL) = 10 ug/mL -> y = 0.05 mL
(50 uL)

Add 100 uL of the appropriate density of effectors to the wells of a

96 well plate

Add 100 uL of the appropriate density of targets to the wells of a 96

well plate

Centrifuge plates for about 30 s at 1000 rpm to promote contact

between effector and target cells.

Incubate plates 3 to 6 hr (this time could vary a lot depending on

the situation (target cells, antigen, T cells, etc.) in a humidified 37
C 5% CO2 incubator.
o Note that effector cells will bind to target cells at room
temperature. Target cell lysis will not begin until the temperature
is increased to 37 C.
o The incubation time can vary markedly depending on the purpose of
the experiment. Under some conditions-e.g., with particularly robust
target cells-overnight incubations are possible.

Continue with the cells with the appropriate cell labeling protocol.