I am trying to analyze the expression of a few molecules in both T CD4+ and T CD8+ lymphocytes from my B16 melanoma tumors (of B6 mice). The issue is that I can't let the tumors grow over 500mm³, or I will get too much necrosis and no live cells to analyze from. But with a tumor of this size, I can get no more than 5x10^6 total cells, which would have to be CD45+ enriched and would leave me with 5x10^4 (TILs are around 1% in B16). After the sorting, I would lose half the cells in the procedure and would end up with 500 cells (CD4+/CD8+ lymphocytes are around 2% of the leukocytes in this model). I need at least 3000ng to do my qPCR and, from my experience, that comes from 8x10^5 leukocytes. If we do the maths, I would need an absurd number of animals.
Has anyone done qPCR from isolated CD8+/CD4+ lymphocytes before? How many animals did you use to get enough sample?
Dear Ana Carolina M. Domingues
Please see attached publication in bioRxiv for single cell transcriptome analysis for TILs (CD8+ T cells) in the B16 melanoma model using 10x Genomics' platform.
I am sure they optimized their cell isolation protocol and their M&Ms will hopefully offer useful advice for your experimental protocol. Following their QC you should be able to analyze gene expression in bulk TILs by qPCR or RNA-seq using your local NGS core facility.
https://www.biorxiv.org/content/10.1101/800847v1.full.pdf
If you have access to a BioAnalyzer (i.e. https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument#0) you will be able to optimize your experimental protocol further as you will get good QC for your isolated RNA.
I hope that helps.
All the best & kind regards,
Michael
Updates: I got about 100.000 cells of each T CD4+ and T CD8+ populations after sorting from enriched CD45+ sample from a pool of three B16 tumors (median size of 400mm³). At the end, around 1ug of RNA from each subset.