Cd11c phycoerythrin cd11b pe

The cell number in each EDTA sample was adjusted to 105 after counting using a Cellometer (Nexcelom Bioscience LLC, USA). After FcBlock (BD Pharmingen, USA) was added at a ratio of 1 : 100, samples were incubated for 10 minutes. Then, samples were mixed and incubated with fluorophore-conjugated antibodies for 20 minutes in the dark. To analyze ATMs, CD45-APC Cy7 (BioLegend, USA), CD68-APC (BioLegend, USA), CD11c-phycoerythrin (CD11b-PE; BioLegend, USA), and CD206-FITC (BioLegend, USA) were used. To analyze KCs, CD45-FITC, F4/80-APC, CD11c-phycoerythrin (CD11b-PE; BioLegend, USA), and CD206-FITC (BioLegend, USA) were used. To analyze T cell population, CD45-FITC (BioLegend, USA), CD3-APC (BioLegend, USA), CD4-PerCp CY5.5 (BioLegend, USA), and CD8-phycoerythrin (BioLegend, USA) were used. Finally, to analyze monocytes, CD45-FITC, CD11b-PerCp CY5.5, and Ly6C-APC (BioLegend, USA) were used. After washing and centrifugation at 1,500 rpm, cells were analyzed using an FACSCalibur (BD Bioscience, USA) instrument and the percentages of the various cell types were determined using the FlowJo program (Tree Star, Inc., USA).

The number of SVCs obtained from the adipose tissue was counted by cellometer (Nexcelom Bioscience LLC, USA), and every sample was set at a concentration of 10⁶ cells. FcBlock (BD Pharmingen, USA) was mixed with the sample at a ratio of 1 : 100, and the reaction was performed for 10 minutes. Fluorophore-conjugated antibodies were added to the shaded state and reacted for 20 minutes. The following antibodies were used: CD45-APC Cy7 (Biolegend, USA), CD68-APC (Biolegend, USA), CD11c-phycoerythrin (CD11b-PE, Biolegend, USA), and CD206-FITC (Biolegend, USA). The samples were analyzed by FACSCalibur (BD Biosciences, USA). A FlowJo (Tree Star, Inc., USA) was used to analyze the percentage of macrophages.

After counting the number of SVCs isolated from the adipose tissue, each sample was modulated to comprise 106 cells using a cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA). FcBlock (BD Pharmingen, USA) was added to the sample at a ratio of 1:100 and allowed to react for 10 min. Fluorophore-conjugated antibodies were added and incubated in the shading state for 20 min. The antibodies used were as follows: CD45-APC Cy7 (BioLegend, USA), F4/80-APC (BioLegend, USA), CD11c-phycoerythrin (CD11b-PE, BioLegend, USA), and CD206-FITC (Biolgend, USA). After washing with 2% FBS/PBS solution and centrifugation at 1500 rpm, the sample was transferred into a fluorescence-activated cell sorting (FACS) tube and analyzed by FACS Calibur (BD Bioscience, USA). Finally, we assessed the percentage of macrophages with CD45(+) F4/80(+), CD45(+)F4/80(+)CD11c(+), and CD45(+)F4/80(+)CD206(+), using FlowJo (Tree Star, Inc., USA).