Does anyone have experience in using absorbance readers (570nm/600nm) in cell titer blue assay (as opposed to the recommended fluorescence)? I am planning on using the Biotek ELX800 for this.
Check out this paper and google CellTiter (one word) to find a brief description of how we used it for absorbance based assays:
http://www.ncbi.nlm.nih.gov/pubmed/23129004
If you can’t download this, let me know and I will post the essential paragraph for you. Generally, fluorescence is more sensitive and gives lower background, but this worked fine for us using absorbance. It depends mainly on your cell culture system. What plate reader or spec you use really doesn’t matter much. We read the absorbance at 490 nm.
Hi Norbert, Thanks for the details. I already had a look over the CellTiter guidelines which indicated that it should be possible to use absorbance instead of fluorescence, allbeit at the loss of some sensitivity.
Why the measuring of absorbance at 490 nm though? Peak absorbance of resazurin and resorufin are 605 and 573 nm respectively, so I thought measuring at 570 would be optimal?
Thank you,
Alec
Pardon my confusion, Alec. When you wrote cell titer, I assumed you were referring to the trademarked CellTiter assay, which uses MTS/formazan (peak absorbance around 490 nm). You seem to be using what is commonly referred to as the Alamar Blue assay. We have used that also (in absorbance mode), and the peak absorbance is around 570 nm. Hope that clears things up.
This is from one of our recent manuscripts (24-well plate method):
Assessment of cell metabolism was performed using the AlamarBlue Cell Viability Reagent (DAL1100, Fisher Scientific, Whitby, Canada) according to the manufacturer’s instructions. Alamar blue (100 µl; 10% of sample volume) was added to each well and the cells were further incubated for 4 h at 37 °C in a humidified 5% CO2 incubator. Absorbance values were recorded at 570 nm.
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