In our laboratory we are trying to determine the toxicity of some compounds against mammal cells, in this case HepG2 cells. So, we have some problems with the Alamar Blue assay, and the culture of HepG2. First, we are growing HepG2 cells with DMEM medium supplemented with 10% FBS and Glutamine. The alamar blue assay was performed with an initially concentration of 10000 cells/well and was incubated with drugs for 48 hours. I’d like to know the time of incubation with resazurin, we have probed 4 and 16 hours, but unfortunately we don’t see any color changes at this time.
Thank you in advance for all the help that you can give me.
Hola Manuel!
Here is a paper that did it in Huh7 cells. At the time the lab was also doing it in Hepa 1-6 (as I did) and HepG2 (I think), and as far as I remember, the protocol was the same.
http://aac.asm.org/content/55/10/4698.full.pdf+html
Hope it helps!
Cat
I evaluated resazurin several years ago for determination of IC50s (growth inhibition) of tumour cell lines such as PC-3, COLO 205, HeLa and MV4-11, but I did not use it for HepG2. Usually adherent cells were attached overnight, then treated with drugs for 72h. For 96-well plates, 10K cells/well of HepG2 is fine, but for 48h treatment, you can add more cells according to the doubling time. Ten (10) uL of resazurin (in PBS) was added to the wells (100 uL of cells with 25 uL of drugs in media), final concentration was about 44 uM, then incubated for 50 to 70min or 120min (MV4-11), read the plates with a microplate reader: EX/EM 530nm/590nm, the fluorescent Signal/background ratio was about 20 to 40. I also used WST-8 method. For IC50 determination, we currently use both SRB and CTG (CellTiter-Glo) methods for about 30 to 40 cell lines including HepG2.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India