Ly6c apc clone al 21

Cells were washed with FACS medium (5% FCS in RPMI) and non-specific binding sites were blocked by incubating 20 minutes at 4°C with an Fc-blocking antibody (anti-CD16/32, clone 2.4G2). Next, cell suspensions were stained with fluorescent conjugated antibodies for 30 minutes at 4°C. Fluorescent antibodies: CD11b PE-Cy7 clone M1/70, F4/80 FITC clone C1:A3-A, Ly6C APC clone AL-21, Ly6G PerCP-Cy5.5 clone 1A8, CD45 APC-Cy7 clone 30-F11, CD4 BV421 clone GK1.5, CD8 BV510 clone 53–67, NK11 PE clone PK136 (BD Biosciences), CD64 Pe clone X54-5/7.1. (BioLegend), CCR2 Pe clone 475301, MerTK Pe clone 108928 (R&D systems), Ly6B clone 7/4 (AbD Serotec)., TCRb APC clone H57-597, CD49b Pecy7 clone DX5, NKp46 PE clone 29A1.4 (eBioscience). Following washing with FACS buffer they were analyzed on a FACS Canto II flow cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star Inc.).

After overnight co-culturing (see above) cells were subjected to flow-cytometrical analysis. Briefly, the cells were washed with FACS medium (5% FCS in RPMI) and non-specific binding sites were blocked by incubating 20 minutes at 4°C with an Fc-blocking antibody (anti-CD16/32, clone 2.4G2). Next, cell suspensions were stained with fluorescent conjugated antibodies for 30 minutes at 4°C. Fluorescent antibodies: CD11b PE-Cy7 clone M1/70, F4/80 FITC clone C1:A3-A, Ly6C APC clone AL-21, Ly6G PerCP-Cy5.5 clone 1A8, CD45 APC-Cy7 clone 30-F11 (BD Biosciences), CD64 PE clone X54–5/7.1. (BioLegend), CCR2 PE clone 475301, MerTK PE clone 108928 (R&D systems), Ly6B clone 7/4 (AbD Serotec). Following washing with FACS buffer they were analyzed on a FACS Canto II flow cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star Inc.).

Cells were centrifuged at 394 g for 7 min and resuspended in FACS medium (5% FCS in PBS) at a concentration of 2.10⁶ cells/ml. Non-specific binding sites were blocked by incubating 20 min. at 4°C with an Fc-blocking antibody (anti-CD16/32, clone 2.4G2). Next, cell suspensions were stained with fluorescent conjugated antibodies for 30 min at 4°C. Fluorescent antibodies: CD11a Pe-Cy7 clone 2D7, CD11b PE-Cy7 clone M1/70, Ly6C APC clone AL-21, CD4 BV421 clone GK1.5, CD8 BV510 clone 53–67, NK11 PE clone PK136, TCRß APC clone H57-597, CD90.2 APC-Cy7 clone Thy1.2, CD138 APC clone 281-2 (BD Biosciences), B220 BV510 clone RA3-6B2, CD93 BV421 clone AA4.1, CD1d PE clone 1B1 and CD49d PE clone 9C10 (eBioscience). Following washing with FACS buffer the cell suspensions were analyzed on a FACS Canto II flow cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star Inc., Ashland, OR). The total number of live 7-AAD- cells in each population was determined by multiplying the percentages of subsets within a series of marker negative or positive gates by the total live cell number determined by microscopy counting with trypan blue for each tissue.