Anti ly6g ly6c

Isolated SVF cells or leukocytes were resuspended in 200 µl PBS containing 0.25% BSA, 0.2 mM EDTA, and 1% penicillin/streptomycin. Cells were preincubated for 7 min at 4 °C in Fc Block (CD16/32, BD Biosciences) and then stained for 15 min with fluorophore-conjugated antibodies at 4 °C. The following antibodies were used: anti-CD45 (clone: 30-F11, Biolegend), anti-F4/80 (clone: BM8, Biolegend), anti-CD11b (clone: M1/70, Biolegend), anti-CD11c (clone: N418, Biolegend), anti-CD206 (clone: C068C2, Biolegend), anti-CCR2 (clone: SA203G11, Biolegend), anti-Ly6G/Ly6C (clone: RB6-8C5, Biolegend), and anti-CD115 (clone: AFS98, Biolegend)^{15 (link)}. Flow cytometric analysis was performed using a FACSCantoII (BD Biosciences). Cell sorting was performed with a FACSAriaII (BD Biosciences). Data were analyzed using FlowJo software (v9.4.10, Tree Star).

The left kidney from each mouse was isolated and transferred to PBS on ice. Kidneys were weighed, cut into small pieces, and digested in a mix of enzymes. Briefly, collagenase type XI (125 U/mL), collagenase type IS (450 U/mL), and hyaluronidase IV-S (60 U/mL) were used for digestion at 37 °C for 20 minutes, with regular agitation. The digested tissue was then passed through a 70 μm sterile cell strainer (Falcon; BD Biosciences, San Jose, CA) to yield a single-cell suspension. Cells were washed and resuspended in fluorescence-activated cell sorting buffer, counted, stained, and collected using multicolor flow cytometry (BD LSR II flow cytometer with DIVA software, BD Biosciences). The macrophage population was defined as CD45+F4/80+CD11b+ cells and further characterized for M1 proinflammatory macrophage (CD11c+) as previously described.^{41 (link)} Following antibodies were used in the panel: anti-CD45, anti-Ly-6G/Ly-6C, anti-CD11b, anti-CD11c (all from BioLegend), and anti-F4/80 (eBioscience). Dead cells were eliminated from analysis using Zombie Violet (BD Biosciences). For each experiment, fluorescence minus one control for each fluorophore was performed to establish gates. In selected experiments, accuracy of the fluorescence minus one gating strategy was confirmed using isotype controls. Data were analyzed by Flow Jo v.10 (Ashland, OR).