Fitc conjugated anti mouse f4 80

The retinas of OIR mice treated with PBS or PDTC were digested in preheated papain solution (Worthington Biochemical Corp., Lakewood, NJ, USA) for 30 minutes. The obtained cell digestion suspension was filtered, centrifuged, and suspended with MACS buffer (BD Biosciences, San Jose, CA, USA). After incubation with anti-mouse CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 20 minutes, these cells were screened with pre-humidified mass spectrometry column (BD Biosciences). Collected CD11b⁺ cells were incubated with FITC-conjugated anti-mouse F4/80 (eBioscience), PE-conjugated anti-mouse CD11c (eBioscience), Alexa Fluor 647-conjugated CD206 (Bio-Rad AbD Serotec, Ltd., Oxford, UK), and the matching control isotype IgG (MCA421; Bio-Rad AbD Serotec) at 4°C for 30 minutes. After washing and resuspension, the cells were analyzed by flow cytometry (BD Biosciences). F4/80⁺/CD11c⁺/CD206^– (F4/80⁺ and CD11c⁺) cells were identified as M1 polarized macrophages, and F4/80⁺/CD11c^–/CD206⁺ (F4/80⁺ and CD206⁺) cells were identified as M2 polarized macrophages as previously described.⁶ (link)^,20 (link)^,21 (link) Data analysis was performed by FlowJo software (BD Life Sciences, Franklin Lakes, NJ, USA).

Subcutaneous tumors were harvested after treatment for a flow cytometer analysis. Single-cell suspensions were prepared by using collagenase/hyaluronidase and DNase digest tumors. Then, the suspensions were lysed with ACK Lysis Buffer to remove red blood cells. Afterwards, cells were incubated with FITC-conjugated anti-mouse F4/80 and PE-conjugated anti-mouse CD86 or PE-conjugated anti-mouse CD206 antibody (eBioscience, CA, USA) and analyzed by flow cytometry. Tumor-associated macrophages (TAMs) exist in two different phenotypes: M1 phenotype was labeled as CD86⁺ in F4/80⁺ and M2 phenotype was labeled as CD206⁺ in F4/80⁺. All the flow cytometric analysis was completed by using FlowJo software.