help... XTT assay - (Nov/03/2006 )
anyone familiar with XTT or done XTT assay before? according to product manual, the more viable cells, the more XTT rings it cleaves and more orange formazan accumulate. therefore when the OD is read, absorbance is higher for samples with more live cells, right? absorbance is lower for the wells with more dead cells right? but my result shows the other way round. the more pathogen protein i put in, the higher the absorbance.
I've tried a quanlitative measure for my experiment using trypan blue. the more pathogen protein i put into my culture, the more cell stained blue (die). the protein i use is total crude protein i got from breaking up a pathogenic bacteria, so it might contain bacteria protein that might trigger cells immune response as well as toxin to kill the cells.
I used a medium with phenol red (although not recommended). at first, i suspect my protein change the pH of medium, thus it changed medium colour. but i also have control wells that contain cells, medium and protein (without XTT), the protein doesn't chnage the colour(pH) of medium. the readings for the wells with different concentration of protein (no XTT) are consistant.
Is the graph i got correct (absorbance increase when pathogen protein concentration is increased)?
this is really weird!
Try doing some control wells with protein, medium and XTT (no cells), maybe for some strange mechanism your protein oxidates XTT to formazan?
The MTT/XTT formazan based assay is a measure of metabolic activity (a mitochondrial assay), not live/dead cells. therefore any decrease in formazan production could be due to more death, cell cycle arrest/senescence, detachment of cells (if you change media between treatment/XTT treatment). Conversly an increased formazan production could mean more cells or an increased metabolic activity of the cells present. Could it be that your pathogen protein is stimulating the cells to require more energy?
ya... sounds logic too. a friend told me that another possibility is the mitochondrial dehydrogenase didn't release from the cells. a friend of my friend got the same result before. he then try to break the cell with DMSO after cells treated with drug/protein, then only he added in XTT to detect the mitochondria product released.
I haven't try that out yet. plan to do it this week after i extract enough protein.
Thanks and wish me good luck!!!