So far we've been using MTT for cell cytotoxicity. We purchased alamar blue from invitrogen newly: how can we use same 96 well plate reading after 1 day,2 day,3 days?
As we know alamar blue is nontoxic so nothing will happen to the cells, but we observed media control giving high fluorescence readings. What can be the reason?
So far I've been using MTT for cell cytotoxicity. As I know alamar blue nontoxic. I think all process should be steril, even plate redaing should be in LAF to use same 96 well plate. In addition , I sent you an assay about alamar blue as an attacment. I hope useful for you.
Article Investigation of the Alamar Blue (resazurin) fluorescent dye...
It seems a nice alternative to MTT. Does anybody already try it?
An alternative to the choices listed above is to measure lactate dehydrogenase (an intracellular enzyme) in your culture medium. If you find LDH in your culture medium your cells are dying. You will need to run a concentration gradient of purified LDH (Sigma) on a fluorometer to detect excitation and emision wavelengths for a standard curve. Then run naive medium as your control and compare cultured medium subtracted by control medium versus your standard curve. You can measure as small as 15 ul in a 96-well fluorescent plate reader, if you run 96-well plates. And measurements can be taken as often as you like. One word of caution, phenol red, which is the pH indicator for most medium interferes with the LDH wavelengths. Hence we switched to Opti-MEM with Glutamax, contains 1/10 amount of phenol red and did not disrupt the LDH assay. Besides allowing us to use the LDH measure on a routine basis Opti-MEM + Glutamax helped the general growth of our cultured adult-derived stem cells as well.
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