Flow cytometric analyses were performed on a FACSCanto flow cytometer (BD Biosciences) as previously described [9 (link),10 ] using the following antibodies: anti-human CD19-APC-H7, anti-human CD14-FITC, anti-human CD79b-APC and anti-human CD16-PE; anti-mouse CD11b-FITC and anti- mouse CD19-PE (all BD Biosciences).
Anti mouse cd11b fitc
Manufactured by BD
Sourced in United States
Anti-mouse CD11b-FITC is a fluorescently labeled antibody that binds to the CD11b antigen expressed on the surface of mouse myeloid cells, including monocytes, macrophages, and granulocytes. It can be used for the identification and enumeration of these cell populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Pe cy7 anti mouse cd86, by BD (2 mentions)
Anti human cd14 fitc, by BD (1 mentions)
Anti human cd19 apc h7, by BD (1 mentions)
Anti human cd16 pe, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Murine ifn γ, by Thermo Fisher Scientific (1 mentions)
12 well plate, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Anti mouse ly6g pe, by BD (1 mentions)
Anti mouse ly6c apc, by BD (1 mentions)
Anti mouse cd206 bv421, by BioLegend (1 mentions)
Perm wash buffer, by BD (1 mentions)
Fixation and permeabilization buffer, by BD (1 mentions)
Anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Pe anti mouse cd11c, by BD (1 mentions)
Anti mouse cd4 pe, by BD (1 mentions)
Anti mouse mhcii pe, by BD (1 mentions)
10 protocols using anti mouse cd11b fitc
1
Flow Cytometric Analysis of Immune Cells
2
Murine Peritoneal Macrophage Activation Assay
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Murine Peritoneal Macrophage cells were seeded in 12-well plate (Thermo Scientific) at density of 1 × 106 per well in 1 mL RPMI 1640 [supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL)] and incubated overnight. Peritoneal macrophage was treated with medium, Pam3CSK4 (InvivoGen, 100 ng/mL) plus murine IFN-γ (40 ng/mL, Peprotech), LPS (InvivoGen, 100 ng/mL) plus murine IFN-γ (40 ng/mL, Peprotech) and indicated SMU-Y6 for 24 h. Cells were collected into centrifuge tube after trypsinization and carefully aspirated the supernatant after centrifugation at 300×g for 5 min. The cells were washed twice with PBS and stained with a mixture of antibodies for 30 min, including anti-mouse CD86-PE-Cy7 (BD Biosciences), anti-mouse CD11b-FITC (BD Biosciences). After incubation, the cells were centrifuged at 300×g for 5 min and washed twice with PBS. Stained cells were analyzed by flow cytometer (FACScanto II, BD) and the flow cytometry data were analyzed using FlowJo software.
3
Isolation and Characterization of Murine Immune Cells
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The epidermis of mice paw was removed and tissue was collected through the scalpel. Mice colon was collected and the contents were removed. Part of colon was separated and washed with PBS three times. The mice paw tissue or colon tissue were placed in a centrifuge tube, RPMI 1640 medium (containing collagenase V, 2 mg/mL) was added in. The tissue was cut into pieces through surgical scissors and was digested at 37 °C for 2 h. The cell suspension was filtered through a 40 μm filter membrane. The cells were collected after centrifugation at 300×g for 5 min. The cells were washed twice with PBS and stained with a mixture of antibodies for 30 min, including anti-mouse Ly6G-APC (Elabscience, E-AB-F1108E), anti-mouse CD11b-FITC (BD Biosciences). After incubation, the cells were centrifuged at 300×g for 5 min and washed twice with PBS. Stained cells were analyzed by flow cytometer (FACScanto II, BD) and the flow cytometry data were analyzed using FlowJo software.
4
MDSC Subset Analysis by Flow
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MDSC induced in vitro from BMC, were collected and stained with fluorescence-labeled antibodies Anti-Mouse CD11b-FITC, Anti-Mouse Ly6C-APC, and Anti-Mouse Ly6G-PE (BD Biosciences, USA), and the proportions of M-MDSC, PMN-MDSC, and total MDSC cells were measured by BD FACSVerse flow cytometer and quantified with FlowJo 7.6 software.
5
MDSC Isolation and Characterization from Bone Marrow Cells
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BMC were isolated from normal mice (on day 0), and cultured with recombinant mouse GM-CSF in complete RPMI 1640 medium for 4 days (on day 4) for defferentiation of MDSC. MDSC induced in vitro from BMC were collected and blocked with Anti-Mouse CD16/32 antibody (Biolegend, USA) for 15 min, and then stained with fluorescence-labeled antibody Anti-Mouse CD11b-FITC (BD Biosciences, USA) or Anti-Mouse CD11b-BV421 (BD Biosciences, USA), in the dark at 4°C for 30 min, followed adding Fixation and Permeabilization buffer (BD Biosciences, USA, 250 μl/tube), at 4°C for 20 min. Cells were washed with 1 ml of 1×Perm/Wash™ buffer (BD Biosciences, USA), and finally, Anti-Mouse CD206-AF647 (BD Biosciences, USA) or Anti-Mouse CD206-BV421 (Biolegend, USA) was added and incubated at 4°C for 30 min in the dark and detected by BD FACSVerse flow cytometer and quantified with FlowJo 7.6 software.
6
Multiparametric Flow Cytometry Analysis
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Cell suspensions obtained from lung were incubated with anti-mouse CD32/CD16 monoclonal antibody (Fc block) for 15 min at 4 °C. Then, cells were incubated with the respective antibody mixes (anti-mouse CD3-FITC, antimouse CD4-PE, anti-mouse CD8-PE, anti-mouse IFN-c-APC, anti-mouse CD11b-FITC, anti-mouse CD11c-PE, anti-mouse IFN-c-PE, anti-mouse MHC-II-PE, anti-mouse IL-10-PE and anti-mouse CD103-biotin, BD PharMingen) for further 30 min at 4 °C and washed with FACS buffer. After staining, cells were acquired on a BD FACSCalibur TM flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software (TreeStar). The total number of cells in each population was determined by multiplying the percentages of subsets within a series of marker negative or positive gates by the total cell number determined for each tissue.
7
Murine AML Cell Line WEHI-3 Culture
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The murine AML cell line, WEHI-3, was maintained in the laboratory of the Department of Clinical Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiao Tong University (Xi'an, China) in DMEM medium supplemented with 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 mg/ml streptomycin; cat no. 15140-122; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The following antibodies were used for flow cytometry (FCM): Fluorescein isothiocyanate (FITC) anti-mouse cluster of differentiation (CD)4 (cat no. 11-0042), allophycocyanin (APC) anti-mouse CD25 (cat no. 17-0251), phycoerythrin (PE) anti-mouse/rat Forheac box P3 (Foxp3) (cat no. 12-5773) and PE anti-mouse CD184 (CXCR4; cat no. 12-9991), and all were purchased from eBioscience (Thermo Fisher Scientific, Inc.). FITC anti-mouse CD11b (cat no. 557396), PE anti-mouse lymphocyte antigen 6G (Ly-6G) and Ly-6C (cat no. 561084) were purchased from BD Biosciences (San Jose, CA, USA). The mouse SDF-1α (cat. no. MCX120), transforming growth factor β (TGF-β; cat. no. MB100B) and interleukin 10 (IL-10; cat. no. M1000B) ELISA kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA), and the arginase 1 (Arg-1; cat. no. JL13668) ELISA kit was purchased from and Shanghai Jiang Lai Biotechnology Co., Ltd. (Shanghai, China; http://www.laibio.com/ ).
8
Comprehensive Murine Immune Cell Profiling
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The following antibodies were used for FACS staining: APC/Cy7 anti-mouse CD3 (100221, Biolegend, San Diego, CA, USA), FITC anti-mouse CD4 (553729, BD Bioscience, San Jose, CA, USA), FITC anti-mouse CD8 (553031, BD Bioscience), APC anti-mouse CD8 (100711, Biolegend), APC anti-mouse Foxp3 (4331294, Invitrogen, Waltham, MA, USA), APC anti-mouse CD62L (104411, Biolegend), PE anti-mouse CD44 (553134, BD Bioscience), APC anti-mouse CD49b (108909, Biolegend), FITC anti-mouse CD11b (557396, BD Bioscience), H-2Kb/OVA (SIINFEKL)-Tetramer/PE was kindly provided by Prof. Eui-Cheol Shin (KAIST, Daejeon, Korea).
9
Multiparametric Flow Cytometry Analysis of Immune Cell Development
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Mouse blood cell and lymphocyte development were analyzed by flow cytometry (36 (link), 37 (link)). B lymphocyte development was conducted by staining with cocktails including FITC anti-mouse CD43 (Biolegend, 553270), PE goat anti-mouse IgM (Southern Biotech, 1020-09), PE-cyanine5 anti-Hu/Mo CD45R (B220) (eBioScience, 15-0452-83), and APC anti-mouse TER119 (Biolegend, 116212). T lymphocyte development was measured on thymocytes using cocktail including PE rat anti-mouse CD4 (BD Pharmingen, 557308), FITC anti-mouse CD8a (Biolegend, 100706), PE/Cy5 anti-mouse CD3e (eBioscience, 15-0031-83), and APC anti-mouse TCRβ (BD Pharmingen, 553174). Myeloid cells were measured in bone marrow cells and splenocytes using cocktail including FITC anti-mouse CD11b (BD Pharmingen, 553310), PE rat anti-mouse CD19 (BD Pharmingen, 557399), PE/Cy5 anti-mouse CD3e (eBioscience, 15-0031-83), and APC anti-mouse Ly6G/Ly6C(Gr-1) (Biolegend, 108412). Purified splenocyte CSR was stained with PE-cyanine5 anti-Hu/Mo CD45R (B220) and FITC rat anti-mouse IgG1(BD Pharmingen, 553443). All antibodies were diluted according to the manufacturer’s protocol. The flow cytometry data were collected on either an LSR II (BD), or on an Attune NxT (Invitrogen) flow cytometer. All flow cytometry data were analyzed using FlowJo V10.
10
Isolation and Flow Cytometry Analysis of Mouse Bone Cells
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The mouse mandibular alveolar bone and femur were obtained according to the above manner. Then the alveolar bone and femur were cut into pieces, digested in 1 mg·mL−1 collagenase I, 1 mg·mL−1 dispase II at 37 °C for 30 min, centrifuged. After lysis of red blood cells on ice, the samples were passed through a 70-μm filter, centrifuged, and ready for staining. FITC anti-mouse Cd11b, PE-Cy7 anti-mouse Cd86, and APC anti-mouse Cd206 were purchased from BD Biosciences, and the permeabilization/fixation kit was purchased from eBioscience. All staining processes were performed in 100 μL PBS. For cell surface staining, after blocking the cell surface Fc receptors, the flow cytometry antibody was directly added to the cell suspension and stained on ice for 30 min. For intracellular antibody staining, the fixed cells were permeabilized and then stained with flow cytometry antibody for 30 min. The samples were then tested using flow cytometry (BD Biosciences), and the flow cytometry data were analyzed and visualized using Flowjo software.
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