C57BL/6 mice (8–10 weeks old) were treated with 35 nmol/kg bryo-1 on days 0, 2, and 3 via IP injection. On day 4, the brains were prepared as described above, and the phagocytosis assay was performed as described (69 (link)). The dissociated cells were resuspended in DMEM/F-12 containing 10% (v/v) FBS in a conical tube, and pHrodo-conjugated myelin (3 μg) was added. The cells were incubated for 4 hours at 37°C, washed sequentially with PBS and PBS containing 2% FBS and 2.5 mM EDTA, and transferred to a 96-well conical-bottom plate. The cells were incubated with TruStain Fc and Zombie NIR (1:1500) for 20 min at RT. The cells were washed, stained with anti-CD11b BV510 (1:400; clone M1/70; Biolegend), anti-CD45 BV605 (1:400; clone 30–11; Biolegend), anti-Ly6G (1:400; clone 1A8; Biolegend), and anti-Clec12a APC (1:400; clone 5D3; Biolegend) antibodies for 30 min at RT, washed twice, and analyzed by flow cytometry on a Cytek Aurora Spectral flow cytometer.
Anti cd11b bv510
Manufactured by BioLegend
Sourced in United States
Anti-CD11b BV510 is a fluorochrome-conjugated monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, and granulocytes. This antibody can be used in flow cytometry applications to identify and characterize these cell populations.
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Lab products found in correlation
Anti cd206 pe, by BioLegend (2 mentions)
Anti cd4 fitc, by BioLegend (2 mentions)
Anti cd45 apc, by BioLegend (2 mentions)
Aurora spectral flow cytometer, by Cytek (1 mentions)
Anti cd45 bv605, by BioLegend (1 mentions)
Anti ly6g, by BioLegend (1 mentions)
Anti f4 80 apc, by BioLegend (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Anti cd45 percp cyanine5, by BioLegend (1 mentions)
Anti cd86 bv605, by BioLegend (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Anti cd45 percp, by BioLegend (1 mentions)
Nir viability dye, by BioLegend (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Anti cd326 fitc, by BioLegend (1 mentions)
Anti ly6g pe cy7, by BioLegend (1 mentions)
Anti cd56 pe, by BioLegend (1 mentions)
Anti cd19 apc, by BioLegend (1 mentions)
Anti cd3 af700, by BioLegend (1 mentions)
Fc blocking antibody, by BD (1 mentions)
7 protocols using anti cd11b bv510
1
Phagocytosis Assay in Bryo-1 Treated Mice
2
Isolation and Characterization of Liver Macrophages
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Mouse livers were perfused with HBSS via the portal vein and followed by 0.04% collagenase IV (C5138, Sigma, MO, USA). Perfused livers were dissected and teased upon 70-μm cell strainers, then suspended in 40 mL DMEM supplemented with 10% FBS. Non-parenchymal cells (NPCs) were separated from hepatocytes by centrifugation at 50 × g for 2 minutes three times. NPCs were plated in cell culture dishes in DMEM supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMax, 100 U/mL penicillin, and 100 mg/mL streptomycin for 15 minutes at 37°C, then the non-adherent cells were removed. The CD45+F4/80+ macrophages adherent cells were used for Flow cytometry. Antibodies used were anti-CD45-PerCP/Cyanine5.5 (103131, Biolegend, CA, USA), anti-CD11b-BV510 (101263, Biolegend, CA, USA), anti-F4/80-APC (123116, Biolegend, CA, USA), anti-CD206-PE (141706, Biolegend, CA, USA) and anti-CD86-BV605 (105037, Biolegend, CA, USA). Cells stained with corresponding isotype control antibodies were used as controls. Data were analyzed using CytExpert software (Beckman, CA, USA).
3
Multicolor Flow Cytometry of Intestinal Epithelial Cells
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Cells were stained with fluorophore-conjugated antibodies for 20 minutes at 4°C. The following antibodies were used for this analysis: anti-CD90.2 BV421, anti-CD11b BV510, anti-CD45 PerCP, anti-Ly6G PE-Cy7, anti-CD326 FITC, anti-CXCR2 PE, and a NIR viability dye (BioLegend, San Diego, CA). The cells were acquired using a FACSCanto flow cytometer (BD Biosciences, San Diego, CA). Epithelial cells were further sorted from live IECs, which were labeled as CD326-positive CD45-negative cells. A FACSAria III flow cytometer (BD Biosciences) was used for sorting. FlowJo software (Tree Star Inc., Ashland, OR) was used for data analysis.
4
Multicolor Flow Cytometry Analysis of PBMCs
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PBMCs were isolated using Ficoll (GE Healthcare, USA) gradient centrifugation, and were suspended in flow cytometry staining buffer (BD Pharmingen, USA) at a final concentration of 107 cells/ml. After incubation with Fixable Viability Stain, BD Horizon™ Brilliant Stain Buffer, and Fc-blocking antibodies (BD Pharmingen, USA), cells were labeled with antibodies specific for surface markers at 4° C for 30 min. Cells were surface-stained with anti-CD4-FITC, anti-CD3-AF700, anti-CD8-Percp.cy5.5, anti-CD11b-BV510, anti-CD14-BV605, anti-CD16-BV421, anti-CD19-APC, anti-CD45-BV650, anti-CD56-PE (Biolegend, USA). To conduct intracellular cytokine staining, the cells were washed, fixed, permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit, and then stained using anti-IL-10-APC-R7 antibodies (BD Pharmingen, USA). The results were analyzed using FlowJo software version 10.0.7.
5
Multiparametric Flow Cytometric Analysis of Immune Cells
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Single cells were incubated with monoclonal anti-CD16/32 (BioLegend, 101302) at 4 °C for 30 min in FACS buffer (DPBS containing 2% FBS) to block the Fc receptors. The cells were then stained with anti-CD45/APC (BioLegend, 103124), anti-CD11b/BV510 (BioLegend, 101263), anti-Ly6G-FITC (BioLegend, 108417), anti-Ly6c-BV421 (BioLegend, 128014), anti-CD11c-PE/cy7 (BioLegend, 117318), anti-MHCII-APC/cy7 (BioLegend, 107628), anti-CD206-PE (BioLegend, 141732), anti-CD63-PE/cy7 (Biolegend, 143910), anti-Spp1-PE (R&D, IC808P), and anti-Fth1-FITC (biobyt, orb102585) in FACS buffer at 4 °C for 30 min, and washed twice with FACS buffer. Intracellular ROS levels were detected by H2DCFDA (Invitrogen, C6827) and Bodipy 493/503 (Invitrogen, D3922) staining. To determine the apoptosis of microglia, flow cytometry analyses were performed using Annexin V-APC apoptosis detection kits (BD, 556547). All antibodies were used at the concentrations recommended by the manufacturer. After being filtered through a round-bottom tube with a 70 μm strainer cap, the cells were analyzed using a BD LSRFortessa™ Cell Analyzer and the FlowJo v10 software (both from BD Biosciences in Fluorescence Core Imaging Center of Ewha Womans University).
6
Multicolor Flow Cytometry for Cell Viability
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Cell pellets obtained from the striatum and midbrain were resuspended in 100 µL of CB and incubated for 5 min at RT with Zombie NIR Dye (BioLegend, San Diego, CA, USA) to assess cell viability (50 µL/sample; 1:2000 dilution in PBS). After quenching with CB, cells were centrifuged at 2000 rpm for 1 min, and labeled with BV510 anti-CD11b (1:500, M1/70, BioLegend), BV421 anti-CD45 (1:1000, 30F11, BioLegend), PE-Cy7 anti-CD8a (1:400, 53 − 6.7, BioLegend) or BUV395 anti-CD8a (1:200, 53 − 6.7, BD Bioscience), and FITC anti-CD4 (1:1000, GK1.5, BioLegend) or BV711 anti-CD4 (1:200, GK1.5, BioLegend) diluted in CB and the FcR Blocking Reagent (1:50; Miltenyi Biotec, Bergisch Gladbach, Germany) during 15 min at 4 °C in the dark. For intracellular staining, cells were incubated with fixation/permeabilization solution (Invitrogen) during 7 min at 4 °C in the dark and washed with PermWASH solution (Invitrogen) and incubated with FITC anti-Ki67 (1:500, 11F6, BioLegend) diluted in PermWASH solution during 15 min at 4 °C in the dark. Once labeled, samples were centrifuged, resuspended in CB and data was acquired using a CytoFLEX LX Flow Cytometer (Beckman Coulter, Brea, CA). The analysis was performed using the CytExpert 2.3 (Beckman Coulter) and the FlowJo 10.0.7r2 (BD Biosciences, Franklin Lakes, NJ) softwares.
7
Flow Cytometry Antibody Panel for Immune Cell Analysis
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Antibodies used for flow cytometry include the following: FITC–anti-CD19 (rat IgG2a; 1:200, #115506; BioLegend), PE–anti-CD45.1 (mouse [A.SW] IgG2a; 1:100, #110707; BioLegend), PE/Cy7–anti-Thy1.1 (mouse IgG1; 1:100, #202518; BioLegend), BV421–anti-CD4 (rat IgG2b; 1:150, #100438; BioLegend), APC–anti-CD45.2 (mouse [SJL] IgG2a; 1:100, #109813; BioLegend), V500–anti-CD3e (Syrian hamster IgG2; 1:50, #560771; BD Biosciences), FITC–anti-CD25 (rat IgG2b; 1:100, #101907; BioLegend), PerCP/Cy5.5–anti-CD19 (rat IgG2a; 1:100, #152405, BioLegend), PerCP/Cy5.5–anti-NK1.1 (mouse IgG2a; 1:100, #108727; BioLegend), PerCP/Cy5.5–anti-CD8b (rat IgG2b; 1:100, #126609; BioLegend), BV605–anti-CD44 (rat IgG2b; 1:100, #103047; BioLegend), FITC–anti-CD8a (rat IgG2a; 1:100, #100705; BioLegend), BV510–anti-CD11b (rat IgG2b; 1:100, #101245; BioLegend), BV421–anti-Ly6C (RçAT IgM; 1:100, #562727; BD Biosciences), and PerCP/Cy5.5–anti-Ly6G (rat IgG2a; 1:100, #127615; BioLegend). For mass cytometry experiments, metal-tagged antibodies were obtained from Fluidigm or conjugated using the Maxpar X8 Antibody Labeling kit according to the manufacturer’s instructions (Fluidigm). Clone and tag information can be found in Table S1.
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