I'm essentially looking for a protocol on how to polarize some RAW 267.4 cell line macrophages. I'm not sure what their phenotype is either, does anybody know of a good protocol to verify the identity of a macrophages (my lab doesn't have access to FACS )
Dear Navin Jayaswal
For the general culture of RAW264.7 macrophages please check the protocols from the Bowdish lab here at McMaster University.
http://www.bowdish.ca/lab/wp-content/uploads/2013/02/Culturing-RAW264.7-Cells.pdf
For the differentiation of RAW264.7 into either M1 or M2 cells please check here.
https://www.spandidos-publications.com/mmr/16/6/9111?text=fulltext
In general you can use either IFNy or LPS for M1 polarization, whereas in contrast to BMDMs IL-4 doesn't seem to potently induce M2 polarization in RAW264.7 cells. For that matter I would suggest to maybe try out IL-10 or following the above mentioned paper an agonistic activation of galectin 9 either directly via antibodies (if possible?) or indirectly via targeting 4-1BB pathway as in the paper below.
http://jem.rupress.org/content/211/7/1433
On a sidenote 4-1BB ligand is highly expressed on RAW264.7 macrophages!
As a readout I would suggest rtPCR (as flow cytometry, which would be my real favourite isn't available to you).
For a collection of M1/M2 markers (albeit in BMDMs that were M1/M2 polarized) please check the following
Article Novel Markers to Delineate Murine M1 and M2 Macrophages
All the best & good luck with your experiment,
Michael