Hi,
I'm using the alamarBlue reagent to continue testing small-molecule drugs on glioblastoma cells. I seed 62500 cells on 96 well plates.
I draw a standard curve first. I go from 500000 cells to 1/2 of 500000 cells and continue 1/2-ing until about 7000 cells and I seed blanks (just media) and I seed in triplicates.
after drawing the standard curve, I use the slope equation to figure out absorbance v cell number.
I got a negative number from the equation... which means there is even less than blank (no cells) I thought of a few reasons:
1) the cells are contaminated, but this I think is unlikely because if there is contamination, that should have also been absorbed and the value should be higher than normal.
2) The media used for the blank is contaminated so the blank absorbance is higher than normal, which I also think is unlikely because the media seems fine. Also I did 3 replicates of this experiment and at some point I used a different media, but I still got negative numbers. Also my supervisor thinks that media contaminated would only cause an insignificant damage but i would see whole chunks of data all being negative numbers
3) The dye reagent is contaminated but for my third replication experiment, I used a newly opened alamarBlue reagent and I still got negative values.
4) I use DMSO to dissolve the drug before treating. So my supervisor told me that I should also treat DMSO (1%) to my standard. But I did not treat DMSO to the blank (Media + dye) could that be a potential cause? if yes, why? I know DMSO is toxic to cells but blanks do not have cells in them... so I don't see how not treating DMSO to the blank is a cause of negative values....
I'm a little worried cuz all three experiments came up with negative values so any help would be appreciated.
thanks!
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