proliferative responses

tylee tylee at itis.com
Mon Sep 25 22:15:09 EST 2000


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 at 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 at 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
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>




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