Non-radioactive Cytotoxicity Assay using Calcein-AM

NK cytotoxicity assay using Calcein-AM

Calcein-acetoxymethyl is a fluoresceinated molecular probe that labels only live cells. It has good retention in targets and low pH sensitivity, and there is no stain transfer among cells. It is a lipid-soluble diester fluorogenic esterase substrate that passively crosses the cell membrane. Once inside the cell it is converted by intracellular esterases into a polar lipid-insoluble fluorescent product (calcein) that is retained by cells with intact membranes but is released by damaged ones, similarly to 51Cr, producing an intense green signal. Calcein-AM is available from Invitrogen.

Target cell preparation
1.   Resuspend targets at 1x106 cells/mL in DMEM-10
2.   Add calcein-AM to final concentration of 15uM and incubate for 30 minutes with occasional mixing
3.   Wash 2x with media and resuspend to 5x104 cells/mL (ie for 5000 targets per well)

Effector cell preparation
1.   Wash cells and resuspend to 2.5x106 cells/mL in DMEM-10
2.   Dispense 100uL media into appropriate numbers of wells (round-bottom) for dilution. Leave first column empty
3.   Dispense 200uL into empty well (50:1), then do serial dilution into next columns of wells to make ratios of 25:1, 12.5:1, 6.25:1, 3.125:1.
4.   Add 100uL of labeled target cells to each well. Have control wells for spontaneous and maximal release.
5.   Add 100uL media to spontaneous release wells
6.   Add 100uL 5% triton X-100 to maximal release wells.
7.   Cover plate, spin cells 5 minutes at 1000 RPM, no brakes
8.   Incubate 4 hours at 37 C
9.   Remove 100uL supernatant and read on plate reader
10.   Excitation wavelength is 485nm, emission is 530nm