Cell Proliferation Kit II (XTT) From Roche

The XTT Cell Proliferation Kit II analyzes the number of viable cells in a culture based on the cleavage of the yellow tetrazolium salt XTT. XTT is cleaved to formazan in viable (metabolically active) cells. The number of viable cells correlates to the amount of orange formazan die. The amount of dye can be recorded on a plate reader by measuring absorbance. This is a colorimetric assay, so a plate reader equipped with a fluorescent bulb is not needed. Any standard plate reader is sufficient.

The kit consists of two reagents: XTT labeling reagent and electron-coupling reagent. Both reagents are stored at -20ºC, but should be aliquoted for individual use to avoid repeated thawing and freezing. (We have found that the XTT reagent can sometimes appear cloudy when it is thawed, but if you let it continue to warm it will clear.) The instruction sheet gives the amount of each that should be aliquoted for 96-well plate assays. Our lab usually uses 24-well plates for the assay, but the amount of each reagent can easily be adjusted for the appropriate final volume required.

The protocol is simple. Once the cells have been cultured under your particular conditions, you can use the kit reagents. The reagents need to be thawed and mixed just before adding the solution to the cells. The cells are then placed back into the incubator for a certain period of time; the incubation period varies with different cell types. At the end of the incubation period, remove the cells and read the absorbance on a plate reader.

Our lab has used four different cell viability assays, and this one is the least complicated and generally the most consistent. Other assays require shaking, multiple additions of reagents, or allowing the cells to cool to room temp before adding reagents. In the case that a particular cell line requires a long incubation period with this assay, it may take more time than other assays. That is one of the few drawbacks. The other potential problem is that we have found that the A549 human cell line does not respond well to this kit. The cells are clearly unhealthy and floating, but still give a similar reading to the healthy cells. Also, human THP-1 cells give inconsistent results if they are undifferentiated. This is certainly not the general rule for this Cell Proliferation Kit II, though, as most cell lines respond well and seem to give results consistent with what is observed through the microscope. We have tried various human cell lines such as HK-2, H441, and HeLa, and various mouse cell lines such as MH-S, RAW 264.7, and J774A.1. All of these cells have very good results with the Cell Proliferation II Kit.

One final benefit is that this assay is non-radioactive so it is safe to perform.