Anti cd45 pacific blue

Single-cell myelin-free suspensions from contralateral and injured regions were plated (5 × 10⁵/96 well), centrifuged, pellet resuspended in 100 μl blocking buffer containing CD16/32 (1:70, Biolegend) and incubated in 150 μl FACs staining buffer containing 2% FBS. For intracellular cytokine staining, cells were fixed (Fixation and Permeabilization kit, BD Bioscience), incubated with antibody mixture on ice for 20 min, washed, centrifuged, resuspended in staining buffer and evaluated on BD LSRII flow cytometer (BD Biosciences). Fluorescence Minus One (FMO) samples, a commonly used strategy to prevent false positive results through overlap of fluorophores [26 (link)], was applied. The following combinations of antibodies diluted 1:200 in FACS staining buffer were used: anti-CD45-Pacific Blue (Biolegend), anti-CD11b-APC-Cy7 (Biolegend), Ly6g (IA8)-AF700 (Biolegend), Ly6c (Hk1.4)-APC (Biolegend), CD206-FITC (Biolegend), IL-10-PE-Cy7 (Biolegend), CD86-FITC (Biolegend), IL-1β-PE (Biolegend). Compensation beads (BD CompBeads) were incubated in Fixation and Permeabilization solution (100 μl, 4 °C, 20 min), incubated with antibody mixture (4 °C, 30 min) and resuspended in staining buffer. Gating and data analysis were performed using FlowJo software (Tree Star).

THP-1 macrophages and bone marrow-derived macrophages were detached using ice-cold PBS, 0.1% EDTA. Lamina propria cells, peritoneal immune cells and lung immune cells were isolated as described previously.^{13 (link),44 (link),45} The cells were then incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min and stained with anti-CD45-Pacific Blue, anti-CD3-BV650, anti-NK1.1-BV650, anti-B220-BV650, anti-CD11b-BV605, anti-CD11c-PECy7, anti-Ly6C-PerCPCy5.5, anti-F4/80-Fitc, anti-CD64-PE, anti-MHC-II-AF700, anti-CD206-PE-TexasRed, anti-pSTAT6-APC (all from BioLegend, mouse cells) anti-CD124-PE, anti-CD206-APC, (all from Biolegend, human cells) for 15–30 min. ZOMBI-NIR live dead stain (BioLegend, San Diego, CA) was used for discrimination between live and dead cells in all experiments. For pSTAT6 staining, cells were permeabilized using the FoxP3 staining kit from eBioscience. Samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc. Ashland, OR). For RNA expression analysis of lung immune cell susbsets, lung homogenates were sorted on a MoFlo Asterios EQ cell sorter (Beckman Coulter Life Sciences, Krefeld, Germany).

Freshly harvested BMNCs were suspended in 4 °C HBSS + (Hanks-Balanced Salt Solution supplemented with 2% FBS, 10 mM HEPES, and 1% penicillin/streptomycin), followed by staining fluorochrome-conjugated or isotype control antibodies on ice for 30 min. The antibodies used in the present study were as follows: anti-CD45-APC (BioLegend, clone 2D1, 1:200), anti-CD45-Pacific Blue (BioLegend, clone 2D1, 1:200), anti-CD31-APC (BioLegend, clone WM59, 1:200), anti-CD31-Pacific Blue (BioLegend, clone WM59, 1:200), anti-CD235a-APC (Biolegend, clone HI264, 1:200), anti-CD235a-Pacific Blue (Biolegend, clone HI264, 1:200), anti-CD43-APC (Biolegend, clone CD43-10G7, 1:200), anti-CD44-PE (Biolegend, clone BJ18, 1:200), anti-CD73-APCcy7 (BioLegend, clone AD2, 1:200), anti-TM4SF1-FITC (Miltenyi Biotec, clone REA851, 1:200), anti-CD118-PE (BD, clone, 1:200), anti-CD140b (PDGFRβ)-APC (BioLegend, clone 18A2, 1:200), and anti-TM4SF1-Alexa Fluor 405 (RandD, FAB8164V, 1:100). Flow cytometry analysis and sorting were performed on a triple-laser MoFlo (Dako) or FACSCalibur (BD) flow cytometer, and data were analyzed using FlowJo software (Tree Star).