Cd68 antibody anti mouse

Mononuclear cells were isolated using Histopaque 1083 (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, using 1.5 mL of peripheral blood diluted 1:1 in PBS. Cells were resuspended in 500 µL of RPMI-1640 for maintenance or cytometry staining.
CD68 antibody anti-mouse coupled to PE-Vio615 (Miltenyi, Bergisch Gladbach, Germany; 130-112-674), CD4 antibody anti-mouse coupled to FITC (Miltenyi, 130-120-750), CD8a antibody anti-mouse coupled to PerCP-Vio700 (Miltenyi, 130-120-756), and CD335 antibody anti-mouse coupled to PE (Miltenyi, 130-112-201) were used for flow cytometry. The BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD, Franklin Lakes, NJ, USA) was used to fix and permeabilize the cells. Washing steps and resuspension for analysis were performed in FACS buffer (PBS/EDTA/FBS).
Flow cytometry was performed according to the manufacturer’s instructions, following Miltenyi’s cell surface flow cytometry staining protocol for CD4, CD8a, and CD335, as well as their intracellular flow cytometry staining protocol for CD68.

After removing red blood cells and dead cells, the isolated single cells were applied to magnetic cell sorting (MACS) according to the method of Korkusuz et al.²⁶ Pdgfra⁺ cells and Pdgfra⁻ cells were separated using magnetic microbeads conjugated with anti‐Pdgfra (CD140a) antibody (CD140a antibody, anti‐mouse, 130‐101‐905, Miltenyi Biotec, Bergisch Gladbach, Germany). Pdgfra⁻/Cd68⁺ and Pdgfra⁻/Cd68⁻ cells were further sorted from Pdgfra⁻ cells using magnetic microbeads conjugated with anti‐CD68 antibody (CD68 antibody, anti‐mouse, 130‐102‐585, Miltenyi Biotec). For single cells isolated from Pdgfra‐mT/mG mice, cells were directly incubated with magnetic microbeads conjugated with anti‐CD68 antibody to separate CD68⁺ and CD68⁻ cells.