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Hello, I isolated and cultured canine PBMC at 10^5 cells/well...

Ana Patricia Ayala Sep 27, 2017

Protocol: In vitro Culture of Human PBMCs

Hello, I isolated and cultured canine PBMC at 10^5 cells/well (in 100 μl) in 96 well plate and added Con A at 10 μg/ml for 48 hours. I did the reading with WST prolif. assay, but the reading show results of less formazan salt formation in my "stimulated" PBMC, compared with my control PBMC Read more more

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Ana Patricia Ayala Answered Sep 27, 2017

Chungbuk National University

I see. I will try proliferation with lower concentrations of Con A. Still the reading would be an accurate reading if I just use WST-1? or is it definitely just accurate with BrdU ELISA or Flow cytometry? Could it be that if I use WST-1 i need to incubate for longer time to have a proliferation reading, since it measures metabolism instead of the DNA synthesis measured through BrdU? As I am very new in the research field, I am truly thankful for your quick reply.

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Balachandran Ravindran Author Answered Sep 27, 2017

Infectious Disease Biology, Institute of Life Sciences, India

Hi The most likely possibility is toxicity of ConA at a concentration of 10ug/ml that you use. Some makes of ConA could be toxic at this concentration. The other possibility is viability of your cells. At the end of 48 hrs you should get >90% viable cells. Hope this helps

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