Anti cd11b apc cy7

Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-coxsackievirus B3 Antibody (MAB948) was purchased from EMD Millipore (Jaffrey, NH). Anti-F4/80 and anti-NFκB p65 were from Abcam (Cambridge, MA). Anti-ICAM-1 and anti-CCR2-PE antibodies were from R&D Systems (Minneapolis MN). Anti-CD16/32 Fc Block, anti-CD11b-APC/CY7, and anti-VLA-4-FITC were from Biolegend. Anti-Gr1-PE-Cy7 was from BD bioscience (San Jose, CA). Alexa Fluor 488, 594 or 633 labeled secondary antibodies were from Life Technologies (Carlsbad, California). The viability dye Ghost violet 510 was from TonBo. For real-time PCR Power SYBR Green RNA-to-Ct^TM 1-Step kit from Thermal Fisher was used.

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).

The antibody catalogue numbers and the concentrations used for immunoblotting (IB), immunocytochemistry (ICC) and flow cytometry (FC) are provided in parentheses. Goat polyclonal anti-Hsp27/Hsp25 (sc-1049; IB 1:1000, ICC 1:100, FC 1:100) was from Santa Cruz Biotechnology. Rabbit polyclonal anti-GFAP (ab7260; IB: 1:10,000, ICC: 1:1000) and β-actin (ab8226; IB: 1:40,000) was from Abcam. Mouse monoclonal anti-α-tubulin (A11126; IB: 1:1000) was from Thermo Fisher Scientific. Rabbit polyclonal anti-Iba1 (019-19741, IB 1:5000, ICC 1:500) was from Wako Laboratory Chemicals and rat polyclonal anti-CD11b-APC/Cy7 (101226, FC 1:1000) was from Biolegend. Mouse monoclonal anti-GFAP-Cy3 (C9205, ICC 1:500, FC 1:500) was from Sigma-Aldrich. Donkey anti-rabbit IgG-AlexaFluor488 (A21206, ICC 1:1000), anti-goat IgG-AlexaFluor488 (A11055, ICC 1:1000, FC 1:1000) secondary antibodies were from Thermo Fisher Scientific. Rabbit anti-mouse IgG-HRP (P0260, IB 1:1000), anti-goat IgG-HRP (P0160, IB 1:2000), and pig anti-rabbit-HRP (P0217, IB 1:2000) secondary antibodies were from Dako-Agilent Technologies.

Macrophages were pre-incubated with LIVE/DEAD Fixable Near-IR cell stain kit (Invitrogen, L34976) and with Mouse BD Fc Block™ (≤ 1 μg/million cells in 100 μL, BD Biosciences #553141) at 4°C for 5 min and then stained with Pacific Blue anti-CD45 (5 µg/mL, BioLegend #109820), anti-CD11b FITC (5 µg/mL, BD Biosciences #557396), anti-CD11b APC-cy7 (2 µg/mL, BioLegend #101226), anti-F4/80 APC (5 µg/mL, BioLegend 123116), or anti-F4/80 PE (2 µg/mL, BD Pharmigen 565410) antibodies. After cells were fixed with formaldehyde 2% and permeabilized with permeabilization buffer (eBioscience, #00-8333-56), ingested erythrocytes were stained intracellularly with anti-TER-119 PE (2 µg/mL, Stemcell #60033) antibody. Stained cells were analyzed using the LSRFortessa (BD) or the SP6800 Spectral Analyzer (Sony). Data were analyzed using FlowJo and FCS express 6 (De Novo software).

Single‐cell suspension from tumors was analyzed by flow cytometry as described earlier [15 (link)]. Briefly, cells were incubated with Fc receptor blocker followed by staining with anti‐CD45‐ Brilliant Violate 421, anti‐F4/80 PerCP/Cy5.5, anti‐CD11b APC/Cy7, anti‐CD206 PE/Cy7, anti‐CCR7 Brilliant Violate 785, and anti‐Gr1 FITC (BioLegend, San Diego, CA, USA). For staining intracellular targets, cells were fixed and permeabilized for 30 min at room temperature (Fixation/Permeabilization Diluent, eBioscience, Waltham, MA, USA). Cells were stained with anti‐EpCAM‐APC (BioLegend). A total of 10 × 10⁶ cells were recorded on a flow cytometer. All the data was recorded on FACS Fortessa (BD Biosciences, Waltham, MA, USA) and analyzed using flowjo software (FlowJo LLC, Ashland, OR, USA).