Hello, I just started to work on human PBMCs to differentiate them into M1 macrophages. I used JOVE's protocol for macrophage differentiation, but I didn't get the results I was expecting as mentioned in the protocol. I seeded 10 million PBMCs into T-25 flask and used RPMI+L-glutamine+ heat inactivated 10%FBS with 50 ng/ml GM-CSF and followed them until day 6. I changed the media on day 3 with a media containing GM-CSF. To dissociate the cells, I used 3 ml 0.25% trypsin-EDTA for 18 mins incubation in 37C, but I couldn't harvest all the cells, and I could get only 8x105 cells. Viability was not affected but I stained the cells with CD3-/CD20-/CD86+/HLA-DR+ to see the cell populations and confirm polarized M1 macrophages population by FACS. Based on this information, my questions are:

1- Is it good to use T-25 for macrophage differentiation or do I need to use different vessel?

2- What is the ideal PBMC amount to seed per cm2? Do I need to see the cell proportions by FACS to optimize my cell density?

3- For detachment, is trypsin the best method for the macrophages, considering that I will be using them for flow cytometry? (30 mins cold PBS on ice and scraping is better?)

4- Is it expected to see a good expression level of CD86 and HLA-DR on the macrophages polarized with GM-CSF induction? Or should I activate them with IFN-g/Poly(I:C)/LPS etc. to see the phenotype by flow cytometry?

Thank you!