FACS analysis of peritoneal lavage cells used the LSR Fortessa 4 (BD Biosciences) and FACS sorting the MOFLO XDP (Beckman Coulter, Inc.), with analysis undertaken using FLowJo vX.0.7 software. Antibodies used were APC anti-mouse CD11b (Cat 101212, clone M1-70, IgG2b, Biolegend), isotype control APC Rat IgG (Cat 400612, clone RTK4530, IgG2b, Biolegend), FITC anti-mouse F4/80 (Cat MCA497FB, clone CI:A3-1, IgG2b, AbD Serotec), and isotype control FITC Rat IgG (Cat 400505, clone RTK2758, Biolegend).
Apc anti mouse cd11b
Manufactured by BioLegend
Sourced in United States
APC anti-mouse CD11b is a monoclonal antibody conjugated to Allophycocyanin (APC) that binds to the CD11b cell surface antigen expressed on mouse myeloid cells, including monocytes, macrophages, and granulocytes.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse f4 80, by BioLegend (4 mentions)
Fitc anti mouse ly 6g, by BioLegend (4 mentions)
Anti mouse cd11c pe, by Thermo Fisher Scientific (3 mentions)
Apc anti mouse cd86, by BioLegend (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Apc cy7 anti mouse ly 6c, by BioLegend (3 mentions)
Facscanto 2, by BD (3 mentions)
Apc cy7 anti mouse f4 80, by BioLegend (2 mentions)
Fitc anti mouse cd80, by Thermo Fisher Scientific (2 mentions)
Pe anti mouse cd11c, by BioLegend (2 mentions)
Fitc anti mouse major histocompatibility complex 2, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse cd40, by Thermo Fisher Scientific (2 mentions)
Pacific blue anti mouse gr 1, by BioLegend (2 mentions)
Pacific blue anti mouse cd4, by BioLegend (2 mentions)
Fitc anti mouse cd8α, by BioLegend (2 mentions)
7 amino actinomycin d viability staining solution, by BioLegend (2 mentions)
Pacific blue anti mouse ly6c, by BioLegend (2 mentions)
Pe cy7 anti mouse ly 6g, by BioLegend (2 mentions)
Lsr fortessa 4, by BD (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
25 protocols using apc anti mouse cd11b
1
Flow Cytometry Analysis of Peritoneal Cells
2
Multicolor Flow Cytometry Analysis
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FACS analysis was performed using routine protocols with a FACS Calibur flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA). Antibodies for staining included APC anti-mouse CD11b (Biolegend, San Diego, CA, USA), Alexa 488 anti-mouse F4/80 (Biolegend), biotin anti-mouse Ly6G (Biolegend) and avidin-PE (eBioscience), PE anti-mouse MR (Biolegend). Dead cells were excluded by propidium iodide staining.
For intracellular staining, cells were stained with cell surface makers first, and then washed with PBS. After centrifuge, cell pellets were suspended completely with fixation buffer on ice for 30 min, and then permeabilized with a Permeabilization Buffer (eBioscience). After centrifuge again, cell pellets were washed and stained with PE anti-iNOS (eBioscience) followed by FACS analysis.
To determine ROS production, cells were harvested and labeled with a ROS probe, according to the recommended protocol (Beyotime), followed by FACS analysis. Briefly, the culture medium was removed and the cells were incubated in dark with the redox-sensitive fluorescent dye (DCFH-DA, 10 µM) diluted by serum-free medium at 37°C/5% CO2 incubator for 30 min. The cells were then washed with PBS and the fluorescent intensity of DCF was detected by FACS. The ROS level was analyzed using the mean fluorescent intensity (MFI).
For intracellular staining, cells were stained with cell surface makers first, and then washed with PBS. After centrifuge, cell pellets were suspended completely with fixation buffer on ice for 30 min, and then permeabilized with a Permeabilization Buffer (eBioscience). After centrifuge again, cell pellets were washed and stained with PE anti-iNOS (eBioscience) followed by FACS analysis.
To determine ROS production, cells were harvested and labeled with a ROS probe, according to the recommended protocol (Beyotime), followed by FACS analysis. Briefly, the culture medium was removed and the cells were incubated in dark with the redox-sensitive fluorescent dye (DCFH-DA, 10 µM) diluted by serum-free medium at 37°C/5% CO2 incubator for 30 min. The cells were then washed with PBS and the fluorescent intensity of DCF was detected by FACS. The ROS level was analyzed using the mean fluorescent intensity (MFI).
3
Multiparametric Flow Cytometry of Murine Liver Cells
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Single cell suspensions of livers were washed in PBS prior to viability staining (Zombie Near-InfraRed) and staining for surface proteins for 30 minutes. Excess antibody was washed out with PBS prior to sample fixation. All antibodies were used at a 1:200 dilution, except F4/80-PE-Cy7 which was used at 1:150, for 30 minutes on ice. Samples were analyzed using a BD LSR-II (4 lasers) and a Cytek Aurora (4 lasers). Antibodies used for flow cytometry included the following: BV421 anti-mouse F4/80 (BD, 100433), eFluor 506 anti-mouse CD45 (Invitrogen, 69-0451-82), FITC anti-mouse Ly6G (BD, 553127), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, 12-0114-81), PE anti-mouse CD11c (Invitrogen, 25-0032-82), PE Cyanine 7 anti-mouse CD3 (Invitrogen, 565411), APC anti-mouse CD11b (Biolegend, 101211) and live/dead near-IR dead cell stain kit (Invitrogen, L34975).
4
Identifying Dendritic Cell Subsets
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Lung single-cell suspensions and BMDC suspensions collected from different groups were stained with PE anti-mouse CD11c (eBioscience, San Diego, CA, USA) and APC-Cy7 anti-mouse F4/80 (Biolegend Inc., San Diego, CA, USA). DCs were marked as CD11c+ and F4/80− cells. This was followed by analysis of the phenotype and the maturation of DCs using FITC anti-mouse CD80, FITC anti-mouse major histocompatibility complex II (MHCII), FITC anti-mouse CD40 (eBioscience, San Diego, CA, USA), and APC anti-mouse CD86 (Biolegend Inc., San Diego, CA, USA) stains. Among them, cDCs were marked as CD11c+MHCII+ double-positive cells. In addition, the cDCs2 (type 2 cDCs) in lung and spleen MNCs were stained as CD11c+CD11b+ double-positive cells using PE anti-mouse CD11c and APC anti-mouse CD11b (Biolegend Inc., San Diego, CA, USA). All the cells, after being washed with PBS, were analyzed using Flow Cytometer (FACSAria™ III, BD Biosciences, USA). The FlowJo software (FlowJo LLC, Ashland, Ore) was used to analyze the data.
5
Murine PBMC Activation by Rickettsial Factors
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Mouse PBMCs (4 × 105 cells) were incubated with culture supernatants of ΔTRP120 or WT-infected or uninfected DH82 cells for 3, 6, and 9 h at 37°C. Harvested PBMCs were resuspended in 100 µL flow cytometry buffer and incubated with anti-mouse CD16/32 Fc blocker (BioLegend), then with APC anti-mouse CD11b (BioLegend) or FITC anti-mouse Ly6C antibodies (BioLegend). Stained cells were fixed and subjected to flow cytometry using the Attune NxT Flow Cytometer System (Thermo Fisher). Data were analyzed in FlowJo software (Ashland, OR).
6
Multiparametric Flow Cytometry Analysis of Immune Cell Subsets
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Cells were pelleted and washed in phosphate-buffered saline (PBS) supplemented with 2% FBS (2% FBS/PBS). The cell suspension was first blocked with TruStain fcX (anti-mouse CD16/32) antibody (BioLegend) for 5 min, and then stained with the following antibodies (Abs) for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, PE anti-mouse F4/80, FITC anti-mouse CD11c, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, Pacific Blue anti-mouse CD4, and FITC anti-mouse CD8α (BioLegend). Next, the cells were washed and resuspended in 2% FBS/PBS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences), and results were analyzed using the FlowJo software (version10.7.0, BD Biosciences).
7
Aortic Cell Isolation and Characterization
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Aortic tissues were minced and digested in digestion solution containing Elastase (Worthington) (0.25 mg ml−1) and LiberaseTH (Roche) (0.025 mg ml−1). Digested tissues were further dissociated with a 21-G needle. Remaining deposited debris was removed and the supernatant was collected after filtering through a 40-μm cell strainer. Cells were suspended in PBS containing 3% FBS, and nonspecific binding of the antibodies to Fc receptors was blocked using an Fc receptor-blocking agent (1:50) (BioLegend). Cells were stained with APC-anti-mouse CD11b (1:150), PE anti-mouse F4/80 (1:20), APC-Cy7 anti-mouse Ly6c (1:300) and Alexa488 anti-mouse CD206 (1:50) (BioLegend). The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used to label dead cells. After washing, cells were analysed using BD FACSVerse. Cell sorting was performed by BD FACSAria II. The data were analysed by Flo Jo software (Tree Star).
8
Comprehensive Immunophenotyping of Murine Immune Cells
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Cells were pelleted, washed with 2% FBS/HBSS, blocked with TruStain fcX (anti-mouse CD16/32) antibodies (BioLegend, CA, USA) for 5 min, and then stained with the following antibodies for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, APC anti-mouse CD3ε, Pacific Blue anti-mouse CD4, PE anti-mouse NK1.1, and FITC anti-mouse CD8α (BioLegend). The cells were then washed and resuspended in 2% FBS/HBSS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed using a BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Data were analyzed using the FlowJo software (version 10.7.0, BD Biosciences). The gating strategy used for flow cytometry analysis was as follows: monocytes (7AAD−CD45+CD11b+Ly-6G−Ly-6Chi), neutrophils (7AAD−CD45+CD11b+Ly-6G+Ly-6Cint), CD4+ T cells (7AAD−CD45+CD3ε+CD4+NK1.1−), CD8+ T cells (7AAD−CD45+CD3ε+CD8α+NK1.1−), and NK cells (7AAD−CD45+CD3ε−NK1.1+) (Figure S1A
9
Etanercept and Metrnl Modulate Immune Responses
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Etanercept (Sigma, Y0001969) was used at 100 uM in vitro and 1 mg/kg in vivo delivery via intraperitoneal injection in 100 uL saline solution. bFGF (Gibco, #13256-029) was used at 10 ng/mL, rMETRNL (R&D Biosystems) was used at 100 ng/mL in vitro and 0.1 ng/dose in vivo. Antibodies used include the following: APC anti-mouse CD45 (1:200, Biolegend 157605), APC anti-mouse CD11b (1:200, Biolegend, 101211), PE anti-mouse CD106 (1:50, VCAM, Biolegend 105713), FITC anti-mouse Ly6A/E (SCA1, 1:50, Biolegend, 108105), Pacific Blue anti-mouse Ly6C (1:200, Biolegend, 128013), AlexaFluoro 488 anti-mouse CD170 (SiglecF, 1:200, Biolegend 155524), AlexaFluoro 488 anti-mouse Ly6G (1:200, Biolegend 127626), APC anti-mouse F4/80 (1:200, Biolegend 123116), Pacific Blue AnnexinV (Biolegend 640926), PE-Cy7 anti-mouse CD11b (1:200, Biolegend 101216). Mouse Meteorin-like DuoSet ELISA (R&D Systems DY6679).
10
Murine Lung Immune Cell Profiling
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On day 10 of PMN mice model, lung tissues were harvested from different treatment groups and mechanically minced and digested to obtain the single-cell-suspensions as described above. The single-cell suspensions washed with PBS and resuspended were incubated with APC-antimouse-CD11b (1:250, Cat. 101211, Biolegend, USA) and PE/Cy7-antimouse-Ly6g (1:150, Cat. 127617,Biolegend, USA) antibodies in 100 μl 1% BSA for 30 min at 4 °C in dark. After centrifuged at 400 × g and washed with PBS, cell pellets were analyzed by BD Fortessa flow cytometry. The data were analyzed using FlowJo software v10.6.2.
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