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

Protocol tips

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Seed 7 x 10^5 cells/mL	Add 2 µM calcein AM and 4 µM EthD-1. Incubate the cells for 30–45 minutes at room temperature

Upstream tips
Seed 7 x 10^5 cells/mL
Protocol tips
Add 2 µM calcein AM and 4 µM EthD-1. Incubate the cells for 30–45 minutes at room temperature

Publication protocol

The experiment was performed on 12- well plates.
The cells before seeding were counted using ADAM NanoEntec ® (7 x 105 cells/mL). To the appropriate wells the cell suspension was poured (200µL cell suspension per well). After 24 hours of incubation at 37 ° C, the pieces of tested materials (1cm x 1cm) were applied to the wells. After 24 hours of incubation with polymers Calcein AM staining was performed. Acetoxymethyl ester of calcein (Calcein AM) is a lipid-soluble diester fluorogenic esterase substrate that passively crosses the cell membrane and is frequently used to stain viable cells. Inside the cells, it is converted by intracellular esterases into a polar, lipid-insoluble fluorescent calcein that is retained by cells with intact membranes but is released from the damaged ones

The cell cultures, after incubation for 24 hours with tested polymers, were rinsed with sterile PBS and to each 1 mLof DMEM without phenol red containing 0,5µL Calcein AM was poured into. After 25 minutes of incubation at room temperaturę, 2mL of DMEM without phenol red was added to each well.
Intensity of calcein fluorescence in live cells was assessed hourly for 24 hours using Multi-Mode Microplate Reader Synergy 2, Biotek®. The fluorescence of calcein was tested on excitation/ emission wavelengths of 485 nm and 528 nm respectively. As a control Calcein AM stained cells seeded on appropriate wells in 12-well plates without tested materials was used. Background signal was subtracted from the results of the experiment.

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