CrReleaseCytotoxicityassay

Description
Cytotoxic activities of mNK cells are examined in 51Cr release assay from target cells 

Procedure
Effector cells (mNK cells) are seeded into round-bottom 96-well plates (Costar) in 100-?l aliquots/well and at appropriate concentrations. Tumor cells, K562 are used as targets. Tumor targets (1 x 106 cells/mL) are incubated with 100 ?Ci sodium chromate (The Radiochemical Centre, Amersham, GB)for 60 min at 37oC, washed and incubated for an additional 30 min to allow spontaneous 51Cr release. Then they are added to the effector cells at the indicated effector to target (E:T) ratios. Incubation is performed for 18h at 37oC in CO2 incubators. All cultures are performed in triplicates and % cytotoxicity is calculated according to the formula: 100 x (test 51Cr release – spontaneous 51Cr release) / (maximum 51Cr release – spontaneous 51Cr release), where maximum and spontaneous counts are calculated from triplicates of target cells 
incubated with 2% Triton X (Sigma) and in plain medium respectively. 

Recipes
Prepare effector cells by a standard "Ficoll-Hypaque" (density 1.077) - procedure. Fill a 50 ml test tube with 15 ml Ficoll solution. Carefully layer the diluted blood sample on top of the Ficoll cushion. Centrifuge for 30 min at 750 x g at room temperature (Notice: without brake otherwise the single cell layer of effector cells will be dirupted). Remove the upper layer and collect the mononuclear cell layer from the interface using a clean
18mm needle. Transfer the cells to a 15 ml test tube. Add 10 ml of PBS, vortex. Centrifuge for 5 min at 250 x g at room temperature. Aspirate the supernatant without
disturbing the cell pellet and resuspend the cell pellet in 1 ml of RPMI 1640 + 10% FCS, determine the cell number in a hemacytometer and adjust the cell concentration to 5 x 106 cells per ml in RPMI 1640 + 10% FCS.
K562 TARGET CELLS is essential to be in exponential phase of growth. Fill a 50 ml test tube with about 20 ml of cell culture. Centrifuge for 5 min at room temperature. Discard the supernatant. Resuspend the cell pellet in 1 ml of CORPMI 1640 + 10% FCS. Determine the cell number and adjust cell concentration to 1 x 105/ml in 1640 + 10% FCS
Add first the effector cells (eg. PBMC, Nk cells etc.)adding in each well the specified number of cells but at a final volume of 100 ?l. Each E:T combination is performed as a triplicate (a typical representation of E:T ratios are: 50:1, 25:1, 12.5:1 etc.). In meantime the target cells are incubated with the radioactive Chromium (100 ?Ci) for 60 min at 37oC in a 15 ml conical tube, washed with PBS and incubated for an additional 30 min to allow spontaneous 51Cr release. The cells are then renumerated and the desired cell number is calculated. Usually 5000 cells/well is an adequate number at a final volume of 100?l/well. Effector cells and target cells are incubated in a final volume of 200 ?l (effector-target cell mixture). Furthermore, triplicates of spontaneous and maximum relase wells are prepared. The spontaneous release wells contain only target cells in the same number as the experimental wells only in culture medium, while the maximum release wells contain target cells in the presence of a chaotropic agent (eg. 2% Triton X (Sigma))which completely destroys all target cells thus releasing the maximum amount of radioactivity in the supernatant. The 96-well plate is incubated for 18h at 37oC in CO2 incubators.
From each well of the 96-well plate 100?l of supernatant are aspired with a pippete carefully not to disrupt the cell pellet, and are added at ?-counter vials. ?-radiation is measured and the counts are used to calculate the cytotoxicity percentage according to the relevant formula (presented above).


Supplies
96-well round bottom tissue culture plates (Costar
T25 Culture flasks
15 ml conical tubes
sodium chromate (51Cr) – 1mCi/ml(The Radiochemical Centre, Amersham, GB)
Triton X (Sigma)
RPMI-1640 culture medium (Life Technologies)
?- radiation counter
18mm fine needle
PBS
Ficoll-Hypaque Solution 
Foetal Calf Serum (FCS; Life Technologies)