Cell culture
Human cell lines used in this study were procured and all cells were grown in Minimal essential medium (MEM, GIBCO) supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal bovine serum (FBS) (growth medium) at 37°C in 5% CO2 incubator.
MTT assay
The MTT assay developed by Mosmann1 was modified and used to determine the inhibitory effects of test compounds on cell growth In Vitro.
The trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue culture plate at a density of 5x103 cells/well in growth medium and cultured at 37°C in 5% CO2 to adhere.
After 48hr incubation, the supernatant was discarded and cells were pretreated with growth medium and were subsequently mixed with different concentrations of test compounds (12.5, 25, 50, 100 and 200 µg/ml) in triplicates to achieve a final volume of 100µl and then cultured for 48 hr.
The compound was prepared as 1.0 mg/ml concentration stock solutions in PBS.
Culture medium and solvent are used as controls.
Each well then added 20µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 4hr at 37°C.
The supernatant growth medium was removed from the wells and replaced with 200 µl of DMSO to solubilize the colored formazan product.
After 30 min incubation, the absorbance (OD) of the culture plate was read at wavelength of 492nm on an ELISA reader.
References:
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 1983, 55.
The MTT assay developed by Mosmann1 was modified and used to determine the inhibitory effects of test compounds on cell growth In Vitro.
The trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue culture plate at a density of 5x103 cells/well in growth medium and cultured at 37°C in 5% CO2 to adhere.
After 48hr incubation, the supernatant was discarded and cells were pretreated with growth medium and were subsequently mixed with different concentrations of test compounds (12.5, 25, 50, 100 and 200 µg/ml) in triplicates to achieve a final volume of 100µl and then cultured for 48 hr.
The compound was prepared as 1.0 mg/ml concentration stock solutions in PBS.
Culture medium and solvent are used as controls.
Each well then added 20µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 4hr at 37°C.
The supernatant growth medium was removed from the wells and replaced with 200 µl of DMSO to solubilize the colored formazan product.
After 30 min incubation, the absorbance (OD) of the culture plate was read at wavelength of 492nm on an ELISA reader.
References:
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 1983, 55.
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