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

protocol 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 Read less more less
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
2 answers
Sort by: most helpful / most recent

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.
0 helpful
0 unhelpful
comment 0 comments down up
share Share

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
0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer