I have converted THP1 monocytes to macrophage using 5 ng/ml PMA for 48 hrs in complete RPMI1640.PMA Containing media was discarded and cells were washed with sterile DPBS for 3 times and given subsequent rest in complete media. mRNA expression of CD11b and CD14 were checked at 48hrs of PMA, 6hrs rest and 24 hrs rest. Both the gene expressions were going down after 6hrs of rest along with TLR4. why it is happening so or it should be like this? Plz help.
Two possible reasons
1- Due to putative induction of macrophage-like cells with pma; CD11b and CD14 might not be stably expressed on these cells... addition of other macrophage inducing agents may help.
2- Expression kinetics of certain surface markers in myeloid cells may differ according to the maturation/differentiation state. Try to correlate mRNA expression with protein levels. Immunophenotyping may also give you certan idea if it is a downregulation in gene expression or excess use of mRNA due to increased translation, or if this mRNA does not translate at all (unstable gene expression).
Lastly, be sure that your RNA samples do not contain gDNA.
Good luck.
CD14 is downregulated on monocytes as they differentiate into macrophages. Additionally, PMA itself can directly result in a decrease in CD14 on monocytes. Similarly, while CD11b is highly expressed on macrophages, the intregin is also decreased on the cell surface by PMA. Both of your findings are consistent with previously published data, so I wouldn't be alarmed. I"ll post links to two papers that might be helpful. Hope this helps!
http://www.ncbi.nlm.nih.gov/pubmed/16228063
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96054/
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques