LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells

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	Add ethidium-calcein mixture, and incubate for 30 min A shorter incubation time may be used if the dye concentrations or incubation temperature are increased.

Protocol tips
Add ethidium-calcein mixture, and incubate for 30 min A shorter incubation time may be used if the dye concentrations or incubation temperature are increased.

Publication protocol

CDC
CHO-K1 and U87MG cells were plated onto 96-well microplates (Costar, Corning) at 30,000 and 20,000 cells/well, respectively, and grown at 37 °C/5% CO2 for 18–24 h. Cells were washed with Hanks' balanced salt solution (BSS) (without phenol red) and incubated at 23 or 37 °C for 60 min with NMO-IgG or NMO-rAb in Hanks' BSS containing 2% pooled normal human complement serum (Innovative Research, Novi, MI) in a final volume of 50 μl. To measure LDH release, the microplate was cooled to room temperature for 10 min, and 50 μl of CytoTox-ONE homogeneous membrane integrity assay solution (Promega, Madison, WI) was added according to the manufacturer's protocol. Complete (100%) LDH release was measured by the addition of 0.5 μl of 1% Triton X-100 to lyse cells. Background (0% lysis) LDH release was measured in cells incubated with complement but no NMO-IgG or NMO-rAb. LDH concentration was assayed from resorufin fluorescence measured on a TECAN Infinite M1000 plate reader (TECAN Groups Ltd., Mannedorf, Switzerland) (excitation/emission 560/590 nm). For live/dead cell staining, cells were washed with Hanks' BSS and then incubated with 1 μm calcein-AM (live cells, green fluorescence) and 2 μm ethidium homodimer-1 (dead cells, red fluorescence) (Invitrogen) in PBS for 15 min prior to imaging.

ADCC
CHO-K1 cells were plated onto 96-well microplates (Costar, Corning) at 20,000 cells/well and grown at 37 °C/5% CO2 for 18–24 h. Cells were then washed with PBS and incubated for 120 min at 37 °C with rAb-53 or rAb-58 (10 and 100 μg/ml), or control-rAb 2B4, and effector NK cells (effector:target ratio 30:1). Cells were washed gently with PBS to remove the remaining NK cells. 1 μm calcein-AM and 2 μm ethidium homodimer-1 were added for 30 min to stain live cells green and dead cells red.

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