2. Chinese Hamster Ovary (CHO) cells
3. 96 well black microtiter plates with clear bottoms (Costar, cat# 3603). Tel: 1-800-4921110
4. PBS (Phosphate buffered saline, 136.8mM NaCl, 2.5mM KCl, 0.8mM Na2PO4, 1.47mM KH2PO4, 0.9mM CaCl2, 0.5mM MgCl2 pH 7.4)
5. 10% Triton X-100 in PBS
6. 0.526mM EDTA in PBS
7. Hams F12 medium Cat# 9058, Fetal Bovine Serum (FBS) Cat# 3000 (Irvine Scientific) Tel: 1-800-437-5706
CHO cells were grown in Hams F12 medium containing 10% FBS. Cells were washed three times with PBS and then treated for 20 minutes with 3 mls of 0.526mM EDTA in PBS. The detached cells were harvested and diluted in PBS. An aliquot was counted using a hemocytometer.
The concentration of cells was adjusted to 2.5 x 105 cells/ml in PBS, which is equivalent to a starting concentration of 2.5 x 104 cells/100l. For preparation of dead cells, half of the cells were treated with 1:100 dilution of 10% Triton X-100 for 10 min. Both live and dead cells were serially diluted 1:3 in PBS to obtain a range of concentrations from 2.5 x 104 to 103 cells/100l. Cells were plated such that each column of the microplate had 8 replicates of a live or dead cell dilution. The negative control/background was 100l of PBS.
A solution of 11.4M cal AM/5.7M EthD-1 in PBS was made and 100l aliquots were added to each well of the microtiter plate, including the control wells. The final concentration of dyes in each well was 5.7M cal AM/2.85M EthD-1. Cells were incubated with the dyes for 30 min at 37oC in a tissue culture incubator and