Phenol red free co2 independent medium

Target U-2 OS or HeLa cells were plated in 24- well glass-bottom imaging dish (MatTek, USA) one day before the imaging experiments. On the day of imaging experiment, primary NK cells were added into the imaging dish at defined NK-to-Target ratio and the two cell types were co-cultured in phenol red-free CO₂-independent medium (Invitrogen) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μl streptomycin and 50 ng/ml or 0.2 ng/ml IL-2. Cell images were acquired using the Nikon TE2000-PFS inverted microscope enclosed in a humidified chamber maintained at 37°C. Cells were imaged every 2, 4 or 10 minutes (varied between experiments), using a motorized stage and a 20X objective (NA = 0.95). To quantify the cumulative survival curves and the phenotype distributions, we viewed and analyzed the images manually, using the MetaMorph software (Molecular Dynamics). Based on morphology, we scored cell death by blebbing followed by cell lysis. The cytotoxic mode was scored based on granzyme-B or caspase-8 specific FRET signal. To quantify the time courses of FRET signal, we used an automatic cell tracking program that we developed using Matlab. The program consists of image analysis procedures that sequentially segment the individual cells, track them in time, as well as measure and ratio the cellular CFP and YFP fluorescence intensity.

Cells were plated in 35 mm imaging dish (μ-dish, ibidi, Germany) and cultured in phenol red-free CO₂-independent medium (Invitrogen) supplemented with 10% FCS, 100 U/ml penicillin and 100 μl streptomycin. Cell images were acquired with the Nikon TE2000-PFS inverted microscope enclosed in a humidified chamber maintained at 37 °C. Cells were imaged every 10 minutes using a motorized stage and a 20X objective (NA = 0.95). Images were viewed and analyzed using the MetaMorph software (Molecular Dynamics).