Whole blood samples from mice were collected and maintained in acid citrate dextrose (Sigma-Aldrich; Merck KGaA). Anti-CD11b eFluor450 (1:100), anti-CD41-allophycocyanin (1:100) (both from eBioscience; Thermo Fisher Scientific, Inc.), anti-Ly6C-PE (1:100; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Ly6G-phycoerythrin (1:100; BD Biosciences) were used to detect leukocyte and platelets antigens. Samples were examined with an LSRII flow cytometer (BD Biosciences) and analyzed using FCS express software (version 3.0, De Novo Software, Glendale, CA, USA). Neutrophils and monocytes were gated through their forward- and side-scatter characteristics and through their Ly-6G+/CD11b+ (neutrophil) and Ly-6G−/CD11b+/Ly-6C+ (monocyte) expression pattern. Platelet-neutrophil and platelet-monocyte aggregates were calculated using CD41 antibody staining.
Anti cd11b efluor450
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Anti-CD11b-eFluor450 is a fluorescent-conjugated 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 product can be used for the identification and analysis of CD11b-expressing cells in flow cytometry applications.
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Lab products found in correlation
Anti cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Anti ly6c apc, by BioLegend (2 mentions)
Acid citrate dextrose, by Merck Group (1 mentions)
Anti ly6c pe, by BD (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Anti cd41 allophycocyanin, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti pd 1 percpcy 5, by BioLegend (1 mentions)
Anti cd3 pe, by Thermo Fisher Scientific (1 mentions)
Anti helios efluor 450, by Thermo Fisher Scientific (1 mentions)
Anti gr 1 pe cy7, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Rmp1 14, by BioXCell (1 mentions)
Anti f4 80 apc, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Anti ly6g pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti ly6c pe, by Thermo Fisher Scientific (1 mentions)
Anti cd3 efluor 450, by Thermo Fisher Scientific (1 mentions)
Anti pd 1 pe cy7, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti cd11b efluor450
1
Murine Leukocyte and Platelet Profiling
2
Investigating Immune Modulation in Tumor-Bearing Mice
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B6 and Rorγ−/− mice were injected i.p. with ID8A-luc cells (4 × 106 cells per mouse). They were treated with either PBS (200 μl i.p.) or RMP1–14 (5 mg kg−1, BioXcell, 200 μl i.p.) on days 3, 6, 9 and 12. Spleens and ascites from untreated B6 and Rorγ−/− mice (n=5 per group) were collected at day 35±2 and cells purified. Cells were stained with Fixable Viability Dye-efluor 780, anti-CD3-PE (6 μg ml−1, eBioscience, clone: 145-2C11), anti-CD4-FITC (15 μg ml−1, eBioscience, clone: RM4-4) and anti-PD1-PerCP-Cy5.5 (6 μg ml−1, BioLegend, clone 29F.1A12). The cells were fixed with Foxp3 Fix/Perm Buffer Set and intracellular staining was performed using anti-Foxp3-APC (6 μg ml−1, eBioscience, clone: FJK-16s) and anti-Helios-efluor 450 (3 μl per 100 μl, eBioscience, clone: 22F6). MDSCs were detected with anti-CD11b-efluor 450 (6 μg ml−1, eBioscience, clone: M1/70) and anti-Gr-1-PE-Cy7 (6 μg ml−1, eBioscience, clone: RB6-8C5).
In addition, Foxp3 mRNA (QT00138369) expression was analysed by TaqMan assay (LightCycler). The expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gadph) mRNA level and expressed as the relative expression, that is, fold increase (2−DCT), where ΔCT=CT(Target gene)−CT(GADPH).
In addition, Foxp3 mRNA (QT00138369) expression was analysed by TaqMan assay (LightCycler). The expression was normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gadph) mRNA level and expressed as the relative expression, that is, fold increase (2−DCT), where ΔCT=CT(Target gene)−CT(GADPH).
3
Multiparametric Flow Cytometry of Tumor Immune Cells
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Tumor tissues from C57BL/6J mice were collected and dissociated into single‐cell suspension by filtering with a 70‐μm mesh cell strainer. Red blood cells were lysed in ACK lysing buffer. Cells were resuspended in Percoll gradients and centrifuged at 400 g for 20 min to isolate lymphocytes. PMA, ionophore, and protein transport inhibitor were added into cell suspension for 5 h incubation. For lymphocyte subpopulations. cells were stained for analysis of surface markers including anti‐CD3 eFluor™ 450 (eBioscience), anti‐CD4 FITC (eBioscience), anti‐CD8 APC (eBioscience), and anti‐PD‐1 PE‐Cy7 (eBioscience), then fixed and permeabilized for anti‐IFN‐γ PE (eBioscience) measurement. For myeloid‐derived suppressor cells (MDSCs) and M2‐like macrophage subpopulations, cells were stained with anti‐CD11b eFluor™ 450 (eBioscience), anti‐Ly6G PE‐Cy7 (eBioscience), anti‐Ly6C PE (eBioscience), anti‐F4/80 APC (eBioscience), and anti‐CD206 FITC (eBioscience). Dead cells were stained with 7‐AAD.
Cultured cells in vitro were stained with PE‐conjugated HLA‐ABC, H‐2Kb, and PD‐L1 (eBioscience) monoclonal antibodies in the dark at 4°C for 1 h, then analyzed using a flow cytometer (BD Bioscience).
Cultured cells in vitro were stained with PE‐conjugated HLA‐ABC, H‐2Kb, and PD‐L1 (eBioscience) monoclonal antibodies in the dark at 4°C for 1 h, then analyzed using a flow cytometer (BD Bioscience).
4
Pristane-Induced Cell Profiling
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Pristane was injected i.p. in WT and CRAMP−/− mice and cells were collected via peritoneal lavage with PBS or after erythrocyte lysis from peripheral blood at day 0 and 7 after injection. Cells were stained extracellularly with anti-CD11b-eFluor450 (eBioscience), anti-Ly6C-APC (BioLegend), anti-CD115-PE (BioLegend), and anti-Ly6G-APC (BioLegend) to determine cell types. Intracellular CRAMP staining was performed with an anti-CRAMP (Innovagen) antibody labelled with an Alexa Fluor 488 antibody labelling kit (LifeTechnologies). Cells were then analyzed by cytofluorometry on a Gallios machine (Beckman Coulter).
5
Multiparameter Flow Cytometry Analysis of Lung Immune Cells
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For flow cytometric analysis of surface markers, single-cell suspensions of lungs were incubated with an anti-FcγRIII/II monoclonal antibody (clone 2.4.G2) and stained with optimal concentrations of the following specific antibodies: anti-CD11b-eFluor450 (Ebioscience, Frankfurt, Germany), anti-CD11b-V500 (clone M1/70; BD Bioscience), anti-F4/80-Alexa-647 (clone BM8; Invitrogen), anti-F4/80-Pacific Blue (clone BM8; Biolegend), anti-Ly6G-PerCp-Cy5.5 (clone1A8; BD Bioscience), and anti-IL-4Rα-PE (clone mIL-4R-M1; BD Biosciences). For intracellular staining of NOS2 and Arg1, single-cell suspensions of lungs were stained for surface markers before permeabilization with Cytofix/Cytoperm® (BD Bioscience). Staining of NOS2 and Arg1 was performed using optimal concentrations of the following specific antibodies: NOS2-FITC (clone 6/iNOS/NOS type II; BD Bioscience), goat-anti mouse Arg1 (clone V-20; Santa Cruz Biotechnology) and polyclonal donkey-anti-goat IgG-Dylight 650 (Abcam, Cambridge, UK). Appropriate isotype controls were used. Fluorescence intensity was measured using a FACSCanto® II flowcytometer (BD Biosciences). Analysis was performed utilizing the FCS Express® program (De Novo Software, Los Angeles, CA, USA).
6
Isolating Mouse PBMC from Blood
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Mouse blood was collected by retro-orbital bleeding into EDTA treated tubes (Microvette, NC0973120). The whole blood volume (∼1 ml per mouse) was then diluted with 1× Dulbecco’s PBS without calcium and magnesium (ratio, 1:1). The diluted samples were subjected to density gradient separation on Ficoll Paque PREMIUM 1.084 (GE Healthcare, GE17-5446-02). After centrifugation, the PBMC layer was collected and washed with red blood cell lysis buffer (Sigma, R7757) to remove remaining red blood cells. PBMC were then washed in 1× PBS and stained for flow cytometric analysis with fluorescently labeled anti–CD45-FITC (eBioscience, clone 30-F11), anti-CD11b-eFluor450 (eBioscience, clone M1/70), anti–Ly6c-PE-Cy7 (Biolegend, clone HK1.4), and anti-Ter119-APC (eBioscience, Ter119) antibodies (at a concentration of 0.2–0.4 µg per 106 cells). Samples were analyzed using a FACSCanto II (BD Biosciences).
7
Fcγ receptor expression on mouse immune cells
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Blood cells of naïve BALB/c WT and Ncf1** mice were analyzed for expression of different Fcγ receptors in steady state or after 2 h of incubation with propidium iodide-labelled SNECs. After hypotonic water lysis cells were stained with anti-CD11b-eFluor450 (eBioscience) and anti-Ly6C-APC (BioLegend), and either anti-CD64-PerCP/Cy5.5, anti-CD16/32-PE/Cy7, or anti-CD16.2-PE (all from BioLegend). Analysis was carried out using a Beckman Coulter Gallios™.
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