Sackler School of
Graduate Biomedical Sciences

CHO cell resistance to Amphotericin B-mediated cell killing

Day 0 Seed 10,000 cells per well in 24-well plates in 0.5 ml of Hams-5%NCS. If analysis will be by Lowry assay, also put medium in an additional 24-well plate for BSA curve.

Day 1 Refeed cells 0.5 ml of Hams-5% LPDS. Do not aspirate media; instead, flick the media into a container in the tissue culture hood.

Day 2 Refeed cells with 0.5 ml of the following media:
Row 1 – H-5% NCS
Row 2 – H-5% LPDS
Row 3 – H-5% LPDS + 20 uM mevinolin + 0.5 mM mevalonate
Row 4 – H-5% LPDS/MM plus 5 ug/ml LDL
Row 5 – H-5% LPDS/MM plus 10 ug/ml LDL
Row 6 – H-5% LPDS/MM plus 20 ug/ml LDL

Day 3 Make amphotericin B (Sigma A-4888) stock solution (10 mg/ml in DMSO). Refeed cells 0.5 ml of Hams-1% LPDS plus 25 ug/ml amphotericin B. Place in CO₂ incubator. After 5 hr, refeed cells 0.5 ml H-5% LPDS. Replace in incubator.

Day 4 Analyze cell survival by protein measurement or crystal violet staining.

Measure protein in wells by Lowry protocol as follows:
Wash 1x, 2 x 5 min, 1x with 1 ml TBS per well. Squirt TBS very gently! Flick TBS into sink. Do not aspirate. To BSA curve plate, add 0, 5, 10, 15, 20, 30 ul BSA (1 mg/ml) in quadruplicate. Add 1 ml Lowry Reagent to each well. Inc on rotator 5 min. Add 50 ul Folin:phenol reagent. Inc on rotator 60 min. Read OD 670 nm.

Concentrations of LDL and amphotericin B may need to be adjusted in preliminary experiments.

Reference: Munn et al. (2003)
Modified September 2006

Return to list of methods