I want to perform a cytotoxicity assay using MCF-7 cells. The kit that I use to determine cell death is colorimetric kit by abcam Cat# ab112118. The protocol says that we need to thaw the kit component A when the incubation time is over and need to add the thawed reagent at 1/5th volume of the assay and incubate at 37 degree in incubator for 1-4 hours followed by absorbance reading.
I am doing this but I am not getting proper results. The value of only media is very high and there is no cytotoxicity seen. I do see some cell death under the microscope. If someone has used this kit, can you please suggest the protocol used?
I came across one publication where they discard the assay media at the end of incubation and make a fresh media with the component and add that to the wells and incubate as mentioned above. If anyone has any suggestions please let me know.
So basically, as I understand properly, you observe elevated absorbance ratio OD570/OD605 only in cells with pure medium, and in all other wells with cells, medium and the compound, you observe no reaction, no change in color?
Perhaps you use to high concentrations of the compound?
Or your compound interacts with the ab112118. Therefore you can indeed, after the incubation time aspirate medium from each well, rinse with buffer three times, add given volume of fresh medium and on top of that add 1/5 volume of ab112118.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence