Jose and Joanne, Just a point of clarification, it is probably not dehydrogenase enzymes that cleaves the tetrazolium salts; but, rather the NADH, NADPH, etc, that is produced by dehydrogenses et al. that reduce the tetrazolium to the formazan form. The tetrazolium powders work well, but I have seen differences among different batches in the amount of background absorbance they have (i.e. without cells present). This is typically controlled for during QC of kit manufacture, so there is an advantage for paying a few dollars more for convenience in some cases. The Promega CellTiter kit you mentioned below comes in different flavors. The MTT version has been around for ~10 years; but there also is an MTS version that is used as a homogeneous assay. The MTS tetrazolium is reduced to a formazan that is aqueous soluble so you do not need to use an organic/detergent solubilization step before you record uding a plate reader. A word of caution that the tetrazolium compounds also can be affected by cell activation because after all, the tetrazolium reduction is a reflection of the cells ability to generate reducing equivalents (i.e. generally equated to level of metabolism). Activated lymphocytes will reduce more tetrazolium than the same number of cells from a quiescent population. I think the same is true for the alamar blue (resazurin -> resorufin) dye. Ty Lee -----Original Message----- From: Joanne Thomas <thomas@tritechinc.com> To: cyto-inbox Date: Wednesday, September 20, 2000 3:39 PM Subject: Re: proliferative responses > >Jose: >You can try MTT, a water soluble tetrazolium salt, which is a yellow >solution that is converted to an insoluble purple formazan when cleaved by >dehydrogenase enzymes. The insoluble formazan can be solubilized isopropanol >and then measured on a spectrophotometer. Sigma sells MTT (Product # M5655) >relatively cheap. In my hands it worked really well for mitogens but was a >bit less sensitive for recall antigens (we used it at 5mg/ml). Some have >used CD69 and flow for this also, correlations were good, however, you'll >have to deal with the issue that cellular activation doesn't always lead to >cellular proliferation. >Joanne >-----Original Message----- >From: Jose Benito <jbenito1@hotmail.com> >To: cyto-inbox >Date: Tuesday, September 19, 2000 6:57 PM >Subject: proliferative responses > > >> >>Hi everyone, I am trying to set up a colorimetric assay to measure >>lymphoproliferative responses to mitogens and recall antigens. I have tried >>the Alamar Blue without any sucess. I will probably try also the celltiter >>assay from promega. Does anyone have experience with this kind of assays? >>Can they really substitute the 3Hthymidine assay? >>Thanks >>Jose Miguel >>Hospital Carlos III >>Madrid >>Spain >>_________________________________________________________________________ >>Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com. >> >>Share information about yourself, create your own public profile at >>http://profiles.msn.com. >> > >
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