BAL cells were incubated for 10 min at 4 °C with an anti-mouse CD16/32 antibody (1:200; Biolegend, San Diego, CA) to block non-specific binding, followed by incubation with a FITC-conjugated anti-mouse CD11b (1:200; Biolegend, San Diego, CA) and an AF 700-conjugated anti-mouse CD11c (1:200, Biolegend) antibodies for 30 min at 4 °C. Data were collected on a BD LSR Fortessa flow cytometer using FACSDiva software (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (Tree Star, San Carlos, CA). A range of 20,000–50,000 cells was analyzed per sample. The initial gating eliminated debris and red blood cells. Next, consecutive gates, were set to identify CD11c+ and CD11b+ cells, as previously described (Swirski et al. 2009 (link); Valdés-Ferrer et al. 2013 (link)). The relative expression of CD11c was used to distinguish CD11c-positive alveolar macrophages (CD11c+, CD11b+) and CD11c-negative infiltrated cells (CD11cneg, CD11b+). Fluorescence minus one (FMO) controls were used for proper gating.
Fitc conjugated anti mouse cd11b
Manufactured by BioLegend
Sourced in United States
FITC-conjugated anti-mouse CD11b is a monoclonal antibody that specifically binds to the CD11b antigen expressed on the surface of mouse myeloid cells, including monocytes, macrophages, and granulocytes. The FITC fluorescent dye is conjugated to the antibody, allowing for the detection and analysis of CD11b-positive cells using flow cytometry.
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Lab products found in correlation
Pe cy7 conjugated anti mouse f4 80, by BioLegend (4 mentions)
Efluor 780 conjugated fixable viability dye, by Thermo Fisher Scientific (4 mentions)
Af 700 conjugated anti mouse cd11c, by BioLegend (3 mentions)
Anti mouse cd16 32, by BioLegend (3 mentions)
Pe conjugated anti mouse ly6c, by BioLegend (3 mentions)
Af 647 conjugated anti mouse ly6g, by BioLegend (3 mentions)
Gallios flow cytometer, by Beckman Coulter (3 mentions)
Apc conjugated anti mouse gr1, by BioLegend (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Anti fsp 1, by Merck Group (1 mentions)
Fitc conjugated secondary antibody, by Abcam (1 mentions)
Pe conjugated anti mouse ly 6g, by BioLegend (1 mentions)
Facs lsr 2, by BD (1 mentions)
Apc conjugated anti mouse ly6g antibody, by BioLegend (1 mentions)
Pe conjugated anti mouse f4 80 antibody, by BioLegend (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
14 protocols using fitc conjugated anti mouse cd11b
1
Multicolor Flow Cytometry Analysis of Alveolar Macrophages
2
Quantification of Myeloid-Derived Suppressor Cells and IL-25-Expressing Fibroblasts
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For detection of MDSCs, cells from mouse lung tissue in each group were collected and stained for 30 min at 4 °C with antibodies against specific cell markers, including FITC-conjugated anti-mouse CD11b (Biolegend; catalogue number: 301403), APC-Cy7 conjugated anti-mouse Ly-6C (Biolegend; catalogue number: 128026) and PE-conjugated anti-mouse Ly-6G (Biolegend; catalogue number: 127618). The percentages of monocytic and granulocytic MDSCs were gated on CD11b+Ly-6C+ cells and CD11b+ Ly-6G+ cells, respectively48 (link). For quantification of IL-25-expressing fibroblasts, individual cells isolated from mouse lung tissue were stained with Alexa Fluor 647-conjugated anti-ER-TR7 (Novus, USA; catalogue number: NB100-64932AF647) and anti-FSP-1 (Millipore; catalogue number: 07-2274) primary antibody followed by FITC-conjugated secondary antibody (Abcam; catalogue number: ab97050). PE-conjugated anti-mouse IL-17E antibody (R&D; catalogue number: IC13991P) was used for staining of intracellular IL-25 molecules in lung tissue. The percentage of IL-25-expressing fibroblasts was gated for FSP-1+ ER-TR7+ cells. Flow cytometry was performed on a FACS LSR II (BD, Netherlands) machine at the Agricultural Biotechnology Research Center (ABRC) in Academia Sinica.
3
Neutrophil Depletion Prior to CLP Procedure
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To deplete neutrophil, mice were treated with intraperitoneal (i.p.) injection of 150 µl (1 mg/kg) anti-Gr-1 antibody (Clone RB6-8C5, BE0075, BioXcell, NH) to deplete neutrophils 24 hours prior to the CLP surgery 38 (link). To verify neutrophil depletion, blood neutrophil levels were assessed by flow cytometry operated on FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA) with APC-conjugated anti-mouse Ly6G antibody (127613, BioLegend, San Diego, California, USA) and FITC-conjugated anti-mouse CD11b (101205, BioLegend, San Diego, California, USA). The data were analyzed with FlowJo V7.6 software (Tree Star, Inc., Ashland, OR).
4
Isolation and Characterization of Aortic Macrophages
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The Mψs, the significant aortic inflammatory cells (4 (link)), were digested and isolated from aortic tissues. Briefly, 1 g of aortic tissues was minced and suspended in 5 ml of Hanks balanced salt solution (HBSS) containing 0.1% (w/v) collagenase type IV (Sigma, United States) for 20 min at 37°C. Next, the specimen was washed with RPMI1640 containing 2% of fetal bovine serum (FBS) and then filtered through a 200-mesh nylon membrane. After centrifugation at 70 × g for 3 min at 4°C, the supernatants were discarded, and the particles were resuspended in 3 ml HBSS. After the erythrocyte lysis, samples were centrifuged for 5 min at 500 × g, 4°C, and then washed two times. The final concentration was adjusted to 1 × 107 cells/ml. Then, 100 μl of suspended cells were stained with KO525-conjugated anti-mouse CD45 (Biolegend, 103137, United States), FITC-conjugated anti-mouse CD11b (Biolegend, 101206, United States), and PE-conjugated anti-mouse F4/80 antibody (Biolegend, 123110, United States). The prepared samples were measured and analyzed using Accuri™ C6 flow cytometer (BD Bioscience, United States).
5
Multicolor Flow Cytometry of Myeloid Cells
6
Flow Cytometry Immunophenotyping Assay
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Single-cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 93; Biolegend, San Diego, CA) for 5 minutes prior to staining for immune cell markers for 15 minutes at room temperature. The following monoclonal antibodies were used: APC-CY7 conjugated anti-mouse CD45 (BD Biosciences Cat:557659 Clone:30-F11), FITC conjugated anti-mouse CD11b (Biolegend Cat:101205 Clone:M1/70), APC conjugated anti-mouse Gr1(Biolegend Cat:108412,Clone: RB6-8C5), FITC conjugated anti-mouse CD8a(Biolegend Cat:100706 Clone:53-6.7), PE conjugated anti-mouseCD4(BD Biosciences Cat:553048 Clone: RM4-5), APC conjugated anti-mouse PD-L1(Biolegend Cat:124311 Clone: 10F.9G2), and IFN-γ (XMG1.2).The flow cytometry analyses were performed using a BD Fortessa Flow Cytometer (BD Fortessa). BD FACS Diva software V.5.0.1 (BD) or Flow Jo (Tree Star) was used for data processed. For cytokine staining, harvested cells were incubated in RPMI-1640 medium with cell activation cocktail with brefeldin A (Bio legend) for 6 hours at 37°C, and stimulated cells were stained as described above.
7
Investigating Peritoneal Inflammation in Mice
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Wild-type or EC-Piezo1-KO mice were injected intraperitoneally with 100 µL PBS containing no or 500 ng TNFα prewarmed to 37°C. After 60 minutes, animals were euthanized, and cells in the peritoneal cavity were collected by flushing with 5 mL ice-cold PBS. Peritoneal cells were filtered using a 70-μm strainer and analyzed by flow cytometry (BD FACS Canto II). The following antibodies were used: FITC conjugated anti-mouse CD11b (101205; BioLegend) and allophycocyanin (APC)-conjugated anti-mouse Ly6G (127614; Biolegend).
8
Quantifying Myeloid-Derived Suppressor Cells in Colon Tumors
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Slides (3–4 µm thick) of colonic tumor specimens were prepared and incubated with FITC-conjugated anti-mouse CD11b (BioLegend, USA, 101205) and APC-conjugated anti-mouse Gr1 (BioLegend, USA, 101211) overnight at 4°C. Images were acquired using a fluorescence microscope (Olympus, Japan). Quantification of fluorescent signals was performed using ImageJ software. The density of infiltrated MDSC in the tumor microenvironment was evaluated by averaged CD11b+Gr1+ co-positive (red and green) area from at least three random 0.42 mm2 fields within the tumors.
9
Quantifying Tumor-Infiltrating MDSCs in FFPE Samples
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For infiltrated MDSCs in tumor tissues, tumor tissues from the mice were fixed in 10% neutralized formalin overnight and embedded in paraffin blocks [formalin fixed paraffin embedded (FFPE)]. FFPE slides that are 3–4 μm thick were cut from the blocks. The slides were deparaffinized with xylene, rehydrated with gradual alcohols, and incubated in 0.01 M of sodium citrate buffer (pH 6.0) in a 95°C water bath for 15 min for antigen retrieval. After being blocked with 5% FBS in 0.1% PBST (Triton X-100–PBS) for non-specific binding sites, the slides were incubated with FITC-conjugated anti-mouse CD11b and PE-conjugated anti-mouse Gr1 (BioLegend, USA) overnight at 4°C. For MDSC density, the CD11b+Gr1+ yellow area in random 0.42-mm2 field within the tumors was measured and averaged. Images were acquired using a fluorescence microscope (Olympus, Japan). Quantification of fluorescent signals was performed using ImageJ software.
10
Multicolor Flow Cytometry Profiling of Lung Immune Cells
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