Pe conjugated anti f4 80

PE-conjugated anti-F4/80 (BD Pharmingen, CA, USA, 565410) was used as a pan-macrophage cell surface marker. To further distinguish macrophage M1 and M2 polarization states, PE-Cy™7-conjugated anti-CD86 (BD Pharmingen, CA, USA, 560582) was chosen to mark the M1 phenotype, and APC-conjugated anti-CD206 (Invitrogen, CA, USA, 17-2061-82) was chosen to mark the M2 phenotype. M1 phenotype was labelled as F4/80⁺CD86⁺, whereas M2 phenotype was labelled as F4/80⁺CD206⁺ (30 (link), 36 (link)).
RAW264.7 cells or macrophages isolated from the spleen of mice were suspended in cold stain buffer (FBS); this was followed by incubation with PE-conjugated anti-F4/80 and PE-Cy™7-conjugated anti-CD86 or APC-conjugated anti-CD206 antibodies on ice in a dark room for 30 min. Then we washed these cells three times with FBS and examined using a CytoFlex flow cytometer (Beckman Coulter, CA, USA) within 2 h. Flow cytometric images were analyzed using CytExpert software. Gating strategy is shown in the Supplementary Figure S2.

For evaluate whether bone marrow-derived macrophages were correctly differentiated, 9 d-cultured cells were stained with anti-macrophage antibodies; PE conjugated anti-F4/80 (BD Bioscience, San Jose, CA) and FITC conjugated anti-CD11b (BioLegend, San Diego, CA). Almost all of the BM-derived cells after 9 days of differentiation were F4/80+CD11b+. (Fig 1A and 1B). A FACScan flow cytometer (BD Biosciences, San Jose, CA) was used to acquire 10,000 events for each sample.

Liver macrophages were isolated from mice and analyzed by flow cytometry as previously described 32 (link). Cells were incubated with PE conjugated Anti- F4/80 (BD Biosciences, USA) and FITC conjugated Anti-CD11b (BD Biosciences, USA), then detected by CytoFLEX flow cytometer (Beckman Coulter, USA), the data were analyzed by CytExpert software (Beckman Coulter, USA). For cell cycle analysis, transfected LX-2 cells were fixed in 70% ethanol at 4 °C for 18 h then incubated with PE. Cell cycle was detected by CytoFLEX flow cytometer (Beckman Coulter, USA). The ratio of cells in G₀/G₁ phase was counted and compared. Apoptosis analysis was detected by Annexin V-FITC/PE staining using FACS Calibur flow cytometer (BD Biosciences, USA), the data were analyzed by FlowJo software (TreeStar, USA).