Cd11b af700

Antibodies were CD45-PE-Cy7 (leukocyte), CD3-AF647 (T cell), CD4-PE (T helper cell), CD25-BV510 and Foxp3-AF488 (regulatory T cell), CD8a-PerCP (cytotoxic T cell) from Biolegend and CD11b-AF700 (phagocyte), CD68-BV510 (pan-macrophage) from AbD serotec and CCR7-AF647 (M1 macrophage), CD206-FITC (M2a macrophage) from Bioss and CD163-PE (M2c macrophage) from LSBio. Single cell suspensions were first stained for the surface markers CD45, CD11b, CD163, CD206, CCR7 (macrophage panel), or CD45, CD3, CD4, CD25, CD8a (T cell panel). Cells were then stained for intracellular staining (CD68) as previously described^{16 (link)}. Data were acquired using Gallios flow cytometer (Beckman Coulter) and fluorescence minus one (FMO) samples were used to set the gates. Evaluation was performed using FlowJo v10 software.

Antibodies were CD45-PE-Cy7, CD3-AF647, CD4-PE, CD25-BV510, Foxp3-AF488, CD8a-PerCP from Biolegend and CD11b-AF700, CD68-BV510 from AbD serotec and CCR7-AF647, CD206-FITC from Bioss and CD163-PE from LSBio. Single cell suspensions were first stained with 50 μl antibody mixture for the surface markers CD45, CD11b, CD163, CD206, CCR7 (macrophage panel), or CD45, CD3, CD4, CD25, CD8 (T cell panel), for 30 minutes in room temperature. Cells were then washed once and incubated with Cytofix/Cytoperm for intracellular staining according to the manufacturer’s instructions (BD Biosciences) at 4 °C for 20 min. Fixed and permeabilized samples were stained with intracellular markers CD68 (for macrophage panel) or Foxp3 (for T cell panel) diluted in Perm/Wash buffer for 30 minutes. Cells were then washed 3 times with Perm/Wash buffer before being fixed in 1% paraformaldehyde. Data were acquired using Gallios Flow Cytometer (Beckman Coulter) and fluorescence minus one (FMO) samples was used to set the gates. Evaluation was performed using FlowJo v10 software.