Killing+Assay

**Killing Assay**

 * E:T ratios (3 replicates)
 * Note that the T cells could come from a 5 day induction or straight from the spleen
 * 100 (100/1=10^6/y -> y = 10^6/100 = 10^4) (10^6 effectors to 10^4 targets)
 * 30 (10^6 effectors to 3.3*10^4 targets)
 * 10 (10^6 effectors to 1*10^5 targets)
 * 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
 * Target cells not pulsed with peptide mixed with effector cells
 * Target cells pulsed with peptide mixed with effector cells that have not been "induced
 * Target cells alone
 * effector cells alone
 * spleen cells and peptide alone
 * other possible controls:
 * Non-enriched T cells (naive and immunized)
 * Plain splenocytes with no 5 day induction (naive and immunized)
 * Determine cells needed
 * Approximate total number of effectors: 1*10^6 * 7 * 3 = 2.1*10^7 cells
 * 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
 * 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.
 * 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
 * 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.
 * 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.
 * 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.