Dear Ebrahim Abdal

It's hard to tell, what exactly went wrong with your experiment. Initially CD68 was cloned in U937 cells, so you should be able to detect expression.

Please see the attached paper:

Article Molecular cloning of CD68, a human macrophage marker related...

There's also a recent publication, where they specifically addressed CD68 expression in monocytic/ myeloid cell (primary/ cell lines), which included U937.

Article Comparative genotypic and phenotypic analysis of human perip...

I would speculate that it is either your antibody, which is not suitable for IHC. For a nice comparison of CD68 expression using different commonly available antibodies in non-myeloid cells, please see:

Article Expression of CD68 in Non-Myeloid Cell Types

For a straightforward phenotypic characterization using flow cytometry, please check:

Article Monocyte Differentiation towards Protumor Activity Does Not ...

I would definitely include positive control, like stimulated (and differentiated) U937 cells that were activated with PMA, PMA+ionomycin or cytokines like IFNg/TNFa/ IL-1b that drive CD68 upregulation.

For a nice general outline for culturing and testing U937 cells, please check:

Article THP-1 and U937 cells

I hope that helps. Good luck with your experiment!

All the best & kind regards,

Michael

CD68 has large intracellular pools.

Did you permeabilize your cells before staining?