Alamar Blue and Lymphocyte Proliferation Assay

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gsskimsing
gsskimsing's picture
Alamar Blue and Lymphocyte Proliferation Assay

Can someone please help!!??  I've been using Alamar Blue to measure the effects of an antagonist on human myeloid and lymphoid cell lines, and been getting good growth curves.  I'm trying to extend the assay to normal human lymphocytes.  Using PHA, I see an increase in AB reduction over 1-3 days post-culture, but get a similar growth curve with the control unstimulated cells, even though under the microscope, you can clearly see blast formation in the PHA cultures.  I tried adding IL-2 as well as trying Con A, but in all cases, the unstimulated cultures show an identical longitudinal increase in AB reduction as unstimulated cultures.  In the old days, 3H thymidine would give me at least 5-10x more cpm between the two.  Any ideas?

marcus muench
marcus muench's picture
 The obvious difference

 The obvious difference between Alamar Blue and 3-H Thymidine is one measures viabilty the other proliferation.  This would suggest that the proliferation that is likely occuring is swamped out by the overall viability of cells in your culture.  In other words, too much noise not enough signal.  You have to play with your conditions so that unstimulated cells die rather than sit there and/or boost overall proliferation.

gsskimsing
gsskimsing's picture
Thanks for that. I got a

Thanks for that. I got a reply from Invitrogen telling me a similar thing. Someone told me that MTS can discriminate between the two, so I'm trying that now.