F4 80 pe

The above isolated cells were blocked with 10 % normal rat serum/PBS on ice for 30 min. The brain mononuclear cells were stained with a combination of anti-mouse antibodies CD206-FITC + MHCII-PE + F4/80-PE/Cy7 + CD45-APC and the splenocytes with a combination of CD11b-FITC + CD45-PE + F4/80-PE/Cy7 + Gr-1-APC or CD206-FITC + MHCII-PE + F4/80-PE/Cy7 + CD45-APC (all from BioLegend) on ice, protected from light, for 30 min. Cells were washed and resuspended in 500 μl PBS/1 % FCS/0.02 % NaN₃. The flow cytometric data were acquired with a two-laser, six-color Gallios flow cytometer and analyzed by Kaluza analysis 1.3 software (Beckman Coulter). Brain mononuclear cells were defined as follows: microglia CD45⁺F4/80⁺, macrophages CD45^hiF4/80⁺, M1-type macrophages CD45^hiF4/80⁺MHCII⁺CD206⁻, and M2-type macrophages CD45^hiF4/80⁺MHCII^−/+CD206⁺. Splenocytes were defined as follows: granulocytes CD45⁺F4/80^−/+Gr-1⁺⁺, monocytes CD45⁺F4/80⁻Gr-1⁺, monocyte-derived macrophages CD45⁺CD11b⁺F4/80⁺Gr-1⁻, tissue-resident macrophages CD45⁺F4/80⁺⁺Gr-1^−/+, M1-type macrophages CD45⁺F4/80⁺MHCII⁺CD206⁻, and M2-type macrophages CD45⁺F4/80⁺MHCII^−/+CD206⁺. Cell populations were calculated as percentages among total leukocytes or macrophages.

Heart single-cell suspension and flow cytometry was prepared as previously described [30 ]. Briefly, hearts were perfused, extracted, finely minced, and then incubated with digestive enzymes at 37 °C on a rocking shaker at 50 rpm for 45–60 min. Samples were homogenized with a 40 μm cell strainer. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Next, samples were centrifuged at 400× g for 5 min at 4 °C, and the pellet was then suspended with FACS buffer. Cells were immune-stained with the following anti-mouse antibodies: CD45-Alexa Fluor^®® 700 (BioLegend, 103128, San Diego, CA, USA), CD11b-PerCP (BioLegend, 101228, CA, USA), F480-PE (BioLegend, 123110, CA, USA), CD206-BV421 (BioLegend, 141717, CA, USA), CD64-APC (BioLegend, 161006, CA, USA), Ly-6G- Brilliant Violet 510™ (BioLegend, 127633, CA, USA), Ly-6C-PE/Cyanine7 (BioLegend, 128018, CA, USA), Propidium iodide (Sigma, 25535-16-4, Darmstadt, Germany). Cells were incubated (30 min, 4 °C) with the antibody mixture in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide) and then washed twice with a staining buffer. Cells were acquired using a LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo V.10 software (FlowJo, Ashland, OR, USA).

Single-cell suspensions from pooled quadriceps and TA muscles were generated as previously described.⁸⁴ The interrogation of live macrophage populations was performed via staining with Zombie NIR viability dye (Cat. #423105; BioLegend, San Diego, CA, USA), CD11b (PerCP-Cy5, Cat. #101228; BioLegend), F4/80 (PE, Cat. #123110; BioLegend), Siglec-F (BV421, Cat# 56268; BD Biosciences, San Jose, CA, USA), Ly6C (FITC, Cat. #128006; BioLegend), CD206 (Alexa Fluor 647, Cat. #141711; BioLegend). Cells were analyzed using a BD FACSAria Fusion flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, version 10; FlowJo, Ashland, OR, USA).