To measure FcγR expression, BMDCs were then stained on ice for 30 min with CD16.2-APC, CD16/32-FITC, CD32-Alexa Fluor 488, or CD64-APC in the absence of Fc-block. The CD16.2, CD16/32, and CD64 antibodies were purchased from BD Bioscience and diluted 1:200. Antibodies were diluted 1:200 in FACS buffer containing HBSS, 1% BSA, 4.17mM sodium bicarbonate, and 3.08mM sodium azide. The CD32 antibody, a gift from Dr. Jeffrey Ravetch (The Rockefeller University, New York, NY), was labeled using an Alexa Fluor 488 Antibody Labeling Kit (Thermo Fisher Scientific). Cells were washed and re-suspended in 2% PFA for analysis on a MACSQuant seven color flow cytometer (Miltenyi Biotech) and data were analyzed using FlowJo Single Cell Analysis Version 7.6.5. To measure pSyk and pErk, cells were stimulated with LPS, oxLDL, oxLDL-Fab2 or oxLDL-IC for 5 or 15min. Cells were then fixed for 10minf in 1× lyse/fix buffer (BD Bioscience) and permeabilized for 30min using Perm Buffer III (BD Bioscience). After permeabilization, cells were Fc blocked for 15min followed by staining with either CD11b-V450 (BD Bioscience), CD11c-FITC (BD Bioscience) and pSyk Y525/526- PE (Cell Signaling Technology); or, CD11b-V450 (BD Biosciences), CD11c-PeCy7 (BD Biosciences) and pERK1/2-FITC (BD Biosciences).
Cd11b v450
Manufactured by BD
Sourced in Germany, United States
CD11b-V450 is a monoclonal antibody that binds to the CD11b antigen, which is expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. This product is intended for use in flow cytometry applications to identify and quantify cells expressing the CD11b marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd11c pe cy7, by BD (2 mentions)
Cd11c fitc, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Ly6g peridinin chlorophyll protein percp cy5, by BD (2 mentions)
Rat anti mouse cd16 32 clone 93, by BioLegend (2 mentions)
Cd45 allophycocyanin apc cy7, by BD (2 mentions)
Lyse fix buffer, by BD (1 mentions)
Perm buffer 3, by BD (1 mentions)
Alexa fluor 488 antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd16 pe cy7, by BD (1 mentions)
Cd64 apc h7, by BD (1 mentions)
Cd54 apc, by BD (1 mentions)
Recombinant murine ifn γ, by Thermo Fisher Scientific (1 mentions)
F4 80 pe, by BD (1 mentions)
Cd86 fitc, by BD (1 mentions)
Apc cy7 anti mouse cd45, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Sphingosine 1 phosphate (s1p), by Merck Group (1 mentions)
10 protocols using cd11b v450
1
Measuring FcγR and Phospho-signaling in BMDCs
2
Multicolor Flow Cytometry Immunophenotyping
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Antibodies CD11b V450, CD16 PE-Cy7, CD18 PE, CD32PerCP-efluor 710, CD54 APC and CD64 APC-H7 were purchased from BD Bioscience (Heidelberg, Germany) and eBioscience (Frankfurt, Germany). As lineage markers, CD66b-FITC (granulocytes) from Beckman Coulter (Krefeld, Germany), CD3-eFluor 450 (T cells) from eBioscience, CD14-APC-Cy7 (monocytes) from BD Bioscience and CD56-BV510 (NK cells) from BioLegend (Fell, Germany) were used. Appropriate isotype antibodies from eBioscience, BD Bioscience and BioLegend were used as controls (see also S1 Table ). Cells were incubated with antibodies for 30 min at 4°C and acquired on a BD FACS Canto II flow cytometer. Analysis and calculations were performed with BD FACS DIVA software.
3
Murine Macrophage Activation Assay
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Sphingosine, sphinganine, S1P, dhS1P, P1P, OVA grade V and LPS were procured from Sigma-Aldrich (St. Louis, MO, USA). Murine recombinant IFN-γ was obtained from PeproTech (London, UK). Antibody against S1PR4 and mouse IL-1β, IL-6, and IL-12b ELISA kit were procured from Boster (Wuhan, Hubei, China). Fluorescent antibody against S1PR4 was obtained from Affinity Biosciences (Cincinnati, OH, USA). Antibodies against iNOS and formyl peptide receptor 2 (FPR2) were purchased from Abcam (Cambridge, MA, USA). Antibodies against p38, p-p38, p65, p-p65, JNK, P-JNK, Erk, p-Erk, FLAG and magnetic beads were procured from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CD68 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Arg-1, IL-1β, and β‐Actin were procured from the Proteintech Group (Wuhan, Hubei, China). Anti-mouse CD45-APC/Cy7, F4/80-PE, CD11b-V450, and CD86-FITC were procured from BD Biosciences (San Jose, CA, USA). Anti-mouse CD206-PE/Cy7 and murine recombinant IL-4 were procured from BioLegend (San Diego, CA, USA). CYM50358 was obtained from Tocris Bioscience (Bristol, UK). All other reagents were obtained from MedChem Express (Monmouth Junction, NJ, USA), except stated otherwise.
4
Quantifying Nasopharyngeal Neutrophils in Pups
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Neutrophils present in the nasopharynx were quantified as previously described (18 (link)). Briefly, nasal lavage samples from individual pups were pelleted by centrifugation at 500 × g for 5 min. Samples were resuspended in 50 μl ACK lysis buffer (Thermo Fisher Scientific) and incubated for 5 min at room temperature. Afterwards, 200 μl of PBS was added to the samples, and cells were pelleted as described above and resuspended in PBS containing 1% bovine serum albumin (BSA). Samples were stained with a LIVE/DEAD Fixable Aqua dead cell stain kit (Invitrogen, Thermo Fisher Scientific). Next, samples were blocked with a 1:200 dilution of a rat anti-mouse CD16/32 (clone 93; BioLegend). Cells were stained for 30 min at 4°C with fluorophore-conjugated antibodies (diluted 1:150) against the following surface markers: CD11b-V450 (BD), Ly6G-peridinin chlorophyll protein (PerCP)-Cy5.5 (BD), and CD45-allophycocyanin (APC)-Cy7 (BD). Samples were run on a BD LSR II flow cytometer and analyzed with BD FACSDiva software.
5
Characterizing Lymph Node Immune Cells
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To analyze immune cell populations in the draining lymph nodes, the cells were isolated by slitting the organs and pressing them through 100 µm mesh cups (Corning, # 52360) to generate single-cell suspensions. 2 × 106 cells were stained with different combination of the following antibodies: CD11c-FITC (# 553801), CD4-PE (# 553730), CD27-PE (# 558754), NK1.1-PE-Cy7 (# 552878), 7AAD (# 559925) CD19-V450, (# 560375, CD11b-V450 (# 560455) all from BD Biosciences; CD3e-APC (# 17–0031) from eBioscience ; AnnexinV-FITC from Immunotools (# 31490013) and CD8a-FITC from Miltenyi (# 130–102–806). A minimum of 5 × 105 events were detected per measurement. Flow cytometry was performed on a FACS Canto II (BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo Software v7.6.5 (Treestar). The gating strategy is representatively depicted in Fig. S2.
6
Quantifying Nasal Neutrophils in Pups
7
Quantifying Neutrophils and Macrophages
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Flow cytometry was performed to quantify the number of neutrophils (CD11b+Ly6G+) and macrophages (CD11b+F4/80+Ly6G–) in each sample. Antibodies used included: Ly6G-APC (Clone: 1A8, BD Bioscience; Cat. #: 560599), CD11b-V450 (Clone: M1/70, BD Bioscience; Cat. #: 560455), F4/80-PECy7 (Clone: BM8, eBioscience; Cat. #: 25-4801-82). No Fc block utilized. Following 1 h of incubation at 4°C, sample were washed and filtered through a 45-micron mesh filter before being run on a FACSAria II (BD Biosciences) Cell Sorter at the University of Michigan Flow Cytometry Core in the Biomedical Science Research Center. Samples were gated to separate debris and autofluorescent signals from the cell population. Data were then analyzed using the FlowJo software (TreeStar). Flow cytometric data was normalized to account for differences in aggregate number between cell types. For untreated group flow data was normalized to the mobile sample population. For all experiments with treated and untreated samples values were normalized to the untreated population.
8
BAL Immune Cell Profiling by Flow Cytometry
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Flow cytometry was used to determine the number and types of cells present in the BAL fluid. Fc-blocked (1 µg/ml; eBiosciences) BAL cells were stained with anti-mouse SiglecF-phycoerythrin (PE; 1 µg/ml; BD Pharmingen), CD45- allophycocyanin (APC; 1 µg/ml; eBiosciences), CD3-PECy5 (1 µg/ml; eBiosciences), CD19-PECy5 (1 µg/ml; eBiosciences), CD11c-PECy7 (1 µg/ml; eBiosciences), MHCII-APC-efluor780 (1 µg/ml; eBiosciences), CD11b-V450 (1 µg/ml; BD Pharmingen), Ly6C-FITC 1 µg/ml; BD Pharmingen) and Ly6G-AlexaFluor700 (1 µg/ml; BD Pharmingen). Fixable Viability Dye eFluor® 506 (eBiosciences) was added to exclude dead cells from the analysis.
9
Isolation and Analysis of Lung Immune Cells
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After euthanasia at 6 days p.i., the lungs were flushed of peripheral circulating cells by pushing 5 ml of PBS through the right ventricle of the heart. Lungs were then removed and placed in RPMI medium plus 10% fetal bovine serum (FBS) on ice. Lung tissues were minced with scalpels into 1- to 2-mm pieces and incubated with 1 U/ml collagenase D (Roche Applied Science, Indianapolis, IN) in RPMI for 30 min at 37°C. Minced pieces were pushed through 100-µm nylon mesh to create a single-cell suspension. Residual red blood cells were lysed with ACK lysis buffer (Sigma-Aldrich, St. Louis, MO). Cells were fixed with 2% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 min in the dark. Approximately 2 × 106 cells were stained the following day with anti-CD16 (FcR block), CD4-phycoerythrin (PE), Ly6G-PE, I-Ad–fluorescein isothiocyanate (FITC), CD11c-FITC, SiglecF-Alexa Fluor 647, CD3-allophycocyanin (APC), CD45-peridinin chlorophyll protein (PerCP)-Cy5.5, CD11c-PE-Cy7, CD19-PE-Cy7, CD11b-V450, NKp46-V450, CD19-APC-Cy7, and CD8-APC-Cy7 (BD Biosciences, eBio, BioLegend, San Diego, CA) for 20 min at 4°C. Cells were analyzed using a BD FACSCanto II instrument (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR).
10
Murine Colonic Lamina Propria Immune Profiling
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Murine colonic lamina propria cells were incubated with LIVE/DEAD® stain [Invitrogen L23105], blocked in CD16/CD32 Fc block [eBioscience 16–0161] before staining with CD45 PerCP-Cy™5.5 [BD 550994], CD11b V450 [BD 560455], CD11c APC [BioLegend 117309], Gr1 PE [BD 553128] [eBioscience 11–4801], and CD3 PE-CyTM7 [BD 560591] antibodies, then fixed in 1% formaldehyde. Cells run on a BD LSR Fortessa [BD Biosciences, Oxford, UK] were analysed using FlowJo 7.6.4 [Tree Star, Inc].
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