I am seeding 1 x 10(5) or 2 x 10(5) in a 24-well plate with DMEM 10% FBS for two days. Polarizing cells with M0, M1 (1ug/ml LPS + 25 U/ml IFNy) and M2 (40ng/ml IL-4) for 24 hours. I have groups with adipose tissue supernatant as well, here not stimulated.
I've noticed that the cells are dropping and don't seem to change morphology.
I also have a low protein yield for Western Blotting
What could be wrong?
Dear Agatha de Assis-Ferreira
I briefly wanted to reach out concerning your problems with your RAW macrophages.
Did you check the following issues:
1. Do you have mycoplasma contamination, which could affect the viability of your cells?
2. Did you make sure that your phenotypic shift is not due to a cell line contamination, misidentification or genetic drift (for which RAW macrophages are known)? A reference vial from ATCC and/or micro-satellite genotyping or re-sequencing may help that?
3. Do you work with RAW macrophages that have been passaged more than 10-30 times that are phenotypically unstable as per:
Article Evaluation of phenotypic and functional stability of RAW 264...
Maybe one or several of these considerations help. For additional input for your differentiation protocol, please consider:
http://www.bowdish.ca/lab/protocols/macrophage-isolation-culture/
All the best & good luck with your experiments,
Michael