Cells were plated at densities of 200,000 cells per well on plastic 6 well tissue culture plates (except controls which were plated at 50,000 cells per well) and left at 37°C to attach.
The solutions were mixed well and left at room temperature for 5 min before the addition of 0.2 mL of concentrated HCl to all the flasks.
After fixation, we added 500 µl of 2M HCl into each well and left at room temperature for one hour.
0.2% SRB (w/v) solution was added after the time for incubation to each well and left at room temperature for 5 20 min.
A volume of 100 μL of 0.057% (w/v) SRB in 1% acetic acid solution was added to each well and left at room temperature for 30 min.
100 μl of substrate [0.1-mg ml-1 3,3',5,5'-tetramethylbenzidine(TMBZ) in 100-mM sodium acetate, pH 6.0, with 0.2% (v/v) of 30% hydrogen peroxide] was added to each well and left at room temperature for blue-color development.
After 4 hours, all the media were removed and 100 μL of 100% DMSO (AppliChem GmbH, Germany) was added into each well and left at room temperature for 30 minutes until the dark blue formazan crystals were dissolved.
One BPB adviser even suggested that if that was all the French could manage, they might just as well leave it at 720p, where it was bound to fail, and save us all the trouble of even considering it.
But a cautionary word before upping the ante – launching into some big rides without taking heed of a few simple rules may well leaving you struggling at the roadside.
"The best that can be said of these topics is that they're so irrelevant no one will ever look at them," she wrote in the post, adding, "If these young scholars are the future of the discipline, I think they can just as well leave their calendars at 1963 and let some legitimate scholars find solutions to the problems of blacks in America.
