CD206 (MMR) Monoclonal Antibody (19.2), PE-Cyanine7, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		All samples were analyzed using FlowJo Version 10 software (Ashland, OR). Forward scatter-area (FSC-A) and side scatter-area (SSC-A) gates were initially set based on size and density using control sample tissues.

Downstream tips
All samples were analyzed using FlowJo Version 10 software (Ashland, OR). Forward scatter-area (FSC-A) and side scatter-area (SSC-A) gates were initially set based on size and density using control sample tissues.

Publication protocol

"Details of the flow cytometry measures are provided in the Supplemental Online Material and Figure S2. All samples were analyzed using FlowJo Version 10 software (Ashland, OR). Forward scatter-area (FSC-A) and side scatter-area (SSC-A) gates were initially set based on size and density using control sample tissues. Within this gated population, a second gate was set using live/dead viability stained cells (Apc-Cy7+) versus FSC-A to exclude apoptotic cells. Both gates were then back-gated to our sample of interest. Within the gated populations on the sample of interest, the singlet cell population was selected using FSC-area vs. FSC-height. We further gated this population of single cells (as identified above) to CD68+ cells (APC-CD68 vs. FSC-Area). Using the CD68+ population, quadrant gating methods were applied to plot for the selected antigens (APC-CD68 vs. PE-CD14, or APC-CD68 vs. PE-Cy7-CD206). The pro-inflammatory or M1 macrophage population was defined as cells dual-stained with the CD68-APC conjugated antibody and CD14-PE conjugated antibody (CD68+/CD14+). The CD206+ macrophages, or M2 population was measured as dual-stained CD68-APC and CD206-PE-Cy7 (CD68+/CD206+). To measure the total CD68+ macrophages we used the number of cells within quadrants which contained the single stained CD68+ cells and the dual stained for either CD14 or CD206.

All of the flow cytometry results are expressed per gram of tissue, taking into account the number of aliquots utilized per sample run. We also report the percentage of ATM’s that are CD14+ and CD206+ and the ratio of CD14+ to CD206+ macrophages."

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