Hi Joelle,

It's definitely a contentious area and there is no perfect answer, we devised the following panel to allow us to both identify and sort M1 and M2.

Marker Dye M1 M2

CD45 BV510 + +

CD11b PE + +

CD11c BV421 - +

CD206 AF488 - +

CD64 AF647 + -

Live/Dead NIR - -

Based on: M2 http://www.nature.com/ijo/journal/v31/n9/full/0803632a.html

M1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944738/

Although, we also considered including CD23, CD36 and CD68 to help further with population discrimination, but decided to limit the panel to a basic setup.

Dear Joёlle!

Please also You look at this article:

https://www.nature.com/articles/s41598-017-08121-8

Flow cytometry

MDMs were stained with monoclonal mouse anti-human CD68, CD80, CD86, CD163, and CD206 (mannose receptor, MR) antibodies (eBioscience, Affymetrix). Cells were suspended in PBS at a concentration of 1 × 105 cells/mL. Non-specific antigens were blocked by 5% BSA buffer. For intracellular CD68, fixation in 4% PFA and permeabilisation with 0.1% Triton X-100 were achieved prior to staining. Cells were analysed using a BD FACSCanto II flow cytometer (BD Biosciences). Non-specific mouse Igs were used as an isotype control.

https://www.jove.com/t/61807/polarization-m1-m2-human-monocyte-derived-cells-analysis-with-flow

Figure 3: Efficacy of the in vitro M1/M2 polarization protocol. Representative dot plots and quadrant gating showing subset frequencies of M1- and M2-polarized cells using CD64 and CD86 (M1) or CD163 and CD200R (M2) in (A) unstained and stained uninfected cells and (B)

Alexandr Chernov this is one of the articles I used and submitted to my supervisor. Based on many readings, I suggested we use CD163, CD86, CD80, CD64, CD206, and I was hesitating on whether or not I should include CCR7. We will also add CD14 to discriminate against dendritic cells, and obviously live/dead.