Hi, guys, I think I meat some problems now. A reviewer asked us to make Western blot to show MAPK and NF-kB pathway upon drug treated Tregs. He said this is doable since it requires only few hundred thousand cells.In addition, he think that from a C57BL/6 mice (splee and LNs) more than 1x10^6 CD4+CD25+ cells can be sorted, which will be more than suffcient to repeat this experiment along with MAPK inhibitors and NF-kappaB inhibitors. However, we can sort only 7*10^5 cells/mice and the maxinium Tregs we can sort is less than 10-15 million and the western blot band for p-p38/p-RelA is too narrow for comparison if these cells are divided into 5 groups. May I know how many Tregs(CD4+CD25+ T cells) could be sorted from 5-10 mice? In addition, how can we operate WB on such rare Tregs in mice body? Thanks
Yours
Shuoyang Liu
In theory, you should get around 1 million CD4+CD25+ cells from a mouse spleen (https://www.stemcell.com/media/files/wallchart/WA10011-Frequencies_Percentages_Mouse_Immune_Cell_Types.pdf). You should have a better yield when pooling cells from multiple mice.
A few Q.
1. What is your sorting strategy? What were your stains and how tight were your gates? If you are losing cells by sorting, try magnetic isolation of Tregs. StemCell Technologies and Miltenyi have kits that you could use. They take less time (meaning your cells would be healthier) and your yields might be better.
2. How many cells did you load into the wells for western blots? We used to lyse 3x10e5 cells in 100 uL and load 25 uL of this and still see good signals for signaling proteins including NFkB and MAPK.
3. Did you probe the membranes for total NFkB and p38? Your total protein bands should be stronger than the phosphorylated bands. It could be that you have very little phosphorylation.
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