The bone marrow and spleen cells were collected from leukemic mice. Red blood cells (RBC) were lysed with NH4Cl RBC lysis buffer for 5 min at room temperature. The nucleated cells were then washed with cold PBS and stained with anti-Gr1-APC (1:20 dilution; cat. no. 553129; BD Biosciences), anti-CD3-PE (1:20 dilution; cat. no. 561824; BD Biosciences) and anti-CD19-PerCP-CyTM5.5 (1:20 dilution; cat. no. 551001; BD Biosciences) for cell lineages, and incubated with anti-CD43-PE (1:20 dilution; cat. no. 553271; BD Biosciences) and anti-B220-APC (1:20 dilution; cat. no. 553092; BD Biosciences) antibodies for B-cell development. The cells were incubated with the antibodies for 20 min at 4°C. Analysis was performed using CytoFLEX flow cytometer and the data were analyzed with CytExpert 2.1 (Beckman Coulter, lnc.).
Anti b220 apc
Manufactured by BD
Sourced in United States
Anti-B220-APC is a fluorescently labeled monoclonal antibody that binds to the B220 antigen expressed on the surface of B cells. It is a tool used in flow cytometry applications for the identification and characterization of B cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 apc, by BD (4 mentions)
Anti cd3 pe, by BD (3 mentions)
Anti cd11b percp cy5, by BD (2 mentions)
Anti mhcii fitc, by BD (2 mentions)
Anti ly6c pe, by BD (2 mentions)
Anti ly6g apc cy7, by BD (2 mentions)
Anti cd45 fitc, by BD (2 mentions)
Anti cd5 pe, by BD (2 mentions)
Anti flk1 pe cy7, by BD (2 mentions)
Anti igm per cp cy5, by BD (2 mentions)
Anti cd11b apc cy7, by BD (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
Anti cd11b pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cd43 pe, by BD (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Anti cd19 fitc, by BD (1 mentions)
Anti cd49b pe, by BD (1 mentions)
11 protocols using anti b220 apc
1
Immunophenotyping of Leukemic Cells
2
Multicolor Flow Cytometry of Murine Immune Cells
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Splenocytes obtained from control or treated tumor bearing mice were liquid-nitrogen frozen and thawed before tests. Then, for lymphoid cell analysis, they were stained in a one-step test with the following fluorophore-labeled anti-mouse monoclonal antibodies (mAbs): anti-CD4-APC (BD Pharmingen, USA, RM4-5), anti-CD8-PE-Cy7 (BD Pharmingen, USA, 53–6.7), anti-CD49b-PE (BD Pharmingen, USA, DX5) and anti-CD19-FITC (BD Pharmingen, USA, 1D3). Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. For myeloid cell characteristics, the flow cytometry analysis of MDSC surface phenotype was performed as described previously [16 (link)] using fluorophore-labeled anti-mouse mAbs: anti-CD11b-PerCP-Cy5.5 (BD Pharmingen, USA, M1/70), anti-B220-APC (BD Pharmingen, USA, RA3-6B2), anti-Ly6G-APC-Cy7 (BD Pharmingen, USA, 1A8), anti-Ly6C-PE (BD Pharmingen, USA, AL-21) and anti-MHCII-FITC (BD Pharmingen, USA, 25-9-17). The cells were stained for 45 min at 4°C. The viability of spleen cells was assessed by incubation with DAPI dye. The analysis was carried out using Becton Dickinson FACSFortessa apparatus with FACSDiva software.
3
Spleen Cell Fractionation and Flow Cytometry
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Spleen cells were stained with anti-CD45-FITC (BD Bioscience, Franklin Lakes, USA), anti-sca1-BV421 (BD Bioscience, Franklin Lakes, USA), anti-flk1-PE-Cy7 (BD Bioscience, Franklin Lakes, USA), anti-B220-APC (BD Bioscience, Franklin Lakes, USA), anti-IgM-Per-CP/Cy5.5 (BD Bioscience, Franklin Lakes, USA), anti-CD11b-APC/Cy7 (BD Bioscience, Franklin Lakes, USA) and anti-CD5-PE (BD Bioscience, Franklin Lakes, USA). Dead cells were identified by using a Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego USA) and doublets were excluded.
The following fractions were sorted by using flow cytometry (FACSAria, BD): CD45+, sca1+/flk1+ (sca1+/flk1+ cells); CD45+, B220+/IgM+, CD11b−/CD5− (B2 cells); CD45+/sca1−/flk1−, B220+/IgM+, CD11b−/CD5− (sca1−/flk1− B2 cells).
The following fractions were sorted by using flow cytometry (FACSAria, BD): CD45+, sca1+/flk1+ (sca1+/flk1+ cells); CD45+, B220+/IgM+, CD11b−/CD5− (B2 cells); CD45+/sca1−/flk1−, B220+/IgM+, CD11b−/CD5− (sca1−/flk1− B2 cells).
4
Isolation and Analysis of Murine Blood, Spleen, and Lung Cells
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20 μl of peripheral blood were collected from the tail vein and collected in FACS buffer. Splenocytes were prepared by grinding spleens between frosted-end microscopic slides in petri dishes containing RPMI medium supplemented with 5% FCS. For analysis of pulmonary cells, mouse lungs were perfused through the pulmonary artery with 5 ml PBS. The lungs were excised, cut into small pieces and incubated for 60 min at 37°C in RPMI medium supplemented with 5% FCS and 200 μg/ml Collagenase D (Roche Diagnostics, Risch, Switzerland) and 10 μg/ml DNAse I (Sigma, Deisenhofen, Germany). The suspension was resuspended with a Pasteur pipette every 15 min. The reaction was stopped by 5 min incubation at 37°C with 5 mM EDTA. Lung cells were then mechanically separated in 100 μm mesh sized cell strainers.
Erythrocytes were lysed with Tris-buffered 0.15 M ammonium chloride for 1 min (lung) or 7 min (blood, spleen). Unspecific binding sites were blocked with anti-FcγR at 4°C for 10 min. The cells were extracellularly stained with anti-CD4-FITC, anti-CD4-APC, anti-B220-APC, anti-CD3-PE, anti-CD8-PerCP-Cy5.5, or anti-CD62L-FITC (all from BD, Heidelberg, Germany), respectively, for 1 h at 4°C. Cells were analyzed on an Accuri C6 cytometer or fixed in 1% PFA for 30 min and analyzed on an LSR-II cytometer (both BD). Absolute cell numbers of blood cell populations were measured with the C6 cytometer.
Erythrocytes were lysed with Tris-buffered 0.15 M ammonium chloride for 1 min (lung) or 7 min (blood, spleen). Unspecific binding sites were blocked with anti-FcγR at 4°C for 10 min. The cells were extracellularly stained with anti-CD4-FITC, anti-CD4-APC, anti-B220-APC, anti-CD3-PE, anti-CD8-PerCP-Cy5.5, or anti-CD62L-FITC (all from BD, Heidelberg, Germany), respectively, for 1 h at 4°C. Cells were analyzed on an Accuri C6 cytometer or fixed in 1% PFA for 30 min and analyzed on an LSR-II cytometer (both BD). Absolute cell numbers of blood cell populations were measured with the C6 cytometer.
5
Multiparametric Flow Cytometry of Tumor Microenvironment
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Spleen and tumor cells obtained from control or treated tumor-bearing mice were thawed, centrifuged, and incubated with monoclonal antibodies conjugated with fluorophores: anti-CD45 V500, anti-CD11b PerCP-Cy5.5, anti-CD11c BV605, anti-CD4 APC, anti-B220 APC, anti-CD49b APC, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHCII FITC, and anti-CD86 PE-Cy7 (all from BD Biosciences). After incubation, cells were suspended in PBS with DAPI dye (Molecular Probes) and analyzed using LSR Fortessa with Diva Software (Becton Dickinson) according to the procedure described by Rossowska and coworkers (27 (link)).
6
Flow Cytometric Analysis of Immune Cell Populations
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For analysis of cellular compositions in thymus, peripheral lymph nodes and spleen in March2−/− and March3−/− mice, the tissues were collected and single-cell suspensions were prepared. After depletion of red blood cells by ammonium-chloride-potassium (ACK) lysing buffer, cells were subjected to stain with the indicated antibodies for 30 min followed by flow cytometry analysis.
For analysis of myeloid cell expansion following Il-3 and Il-34 stimulation, cells were subjected to stain with the indicated antibodies for 30 min and analyzed by flow cytometry.
The following antibodies were used for flow cytometric analyses: anti-CD115-APC (eBioscience, 17-1152-82); anti-CD11b-FITC (BioLegend, 101206); anti-F4/80-PE (eBioscience, 12-4801-82); anti-CD3-FITC (BD Biosciences, 561798); anti-CD4-PE (BD Biosciences, 553048); anti-CD4-APC (BD Biosciences, 553051); anti-CD8-PB (BD Biosciences, 558106); anti-B220-APC (BD Biosciences, 553092); anti-F4/80-APC (BioLegend, 123115); anti-Ly6C-PE/Cy7 (BioLegend, 128017). Data were acquired on a Fortessa™ X-20 (BD Biosciences) and analyzed with FlowJo software.
For analysis of myeloid cell expansion following Il-3 and Il-34 stimulation, cells were subjected to stain with the indicated antibodies for 30 min and analyzed by flow cytometry.
The following antibodies were used for flow cytometric analyses: anti-CD115-APC (eBioscience, 17-1152-82); anti-CD11b-FITC (BioLegend, 101206); anti-F4/80-PE (eBioscience, 12-4801-82); anti-CD3-FITC (BD Biosciences, 561798); anti-CD4-PE (BD Biosciences, 553048); anti-CD4-APC (BD Biosciences, 553051); anti-CD8-PB (BD Biosciences, 558106); anti-B220-APC (BD Biosciences, 553092); anti-F4/80-APC (BioLegend, 123115); anti-Ly6C-PE/Cy7 (BioLegend, 128017). Data were acquired on a Fortessa™ X-20 (BD Biosciences) and analyzed with FlowJo software.
7
Comprehensive Immunophenotyping by Flow Cytometry
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Flow cytometry analysis was performed as previously described [28 (link), 30 (link)]. Samples were measured on a FACSCalibur or FACSCanto II (BD Bioscience, Franklin Lakes, USA) and analyzed with FlowJo (Tree Star, Ashland, USA). Dead cells were identified by using a Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego USA). Doublets were excluded in every analysis. Samples were stained with anti-sca1-APC (R&D Systems, Minneapolis, USA) anti-flk-1-PE (BD Bioscience, Franklin Lakes, USA), anti-B220-APC (BD Bioscience, Franklin Lakes, USA), anti-IgM-Per-CP/Cy5.5 (BD Bioscience, Franklin Lakes, USA), anti-CD11b-APC/Cy7 (BD Bioscience, Franklin Lakes, USA), anti-CD5-PE (BD Bioscience, Franklin Lakes, USA), anti-sca1-FITC (BD Bioscience, Franklin Lakes, USA), anti-flk1-PE-Cy7 (BD Bioscience, Franklin Lakes, USA), anti-CD3-FITC (BD Bioscience, Franklin Lakes, USA), anti-CD4-PE/Cy7 (eBioscience, Waltham, USA), anti-CD8-APC (Biolegend, San Diego, USA), anti-sca1-PerCP/Cy5.5 (eBioscience, Waltham, USA), anti-CD11b-PE (BD Bioscience, Franklin Lakes, USA), anti-CD115-APC (Biolegend, San Diego USA), anti-Gr1-FITC (BD Bioscience, Franklin Lakes, USA) and anti-CD45 FITC (BD Bioscience, Franklin Lakes, USA).
8
Mouse Spleen Cell Isolation and BrdU Incorporation Analysis
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Mononuclear cells from mouse spleens or other organs were extracted using a cell strainer and stained with anti-B220–APC and anti-CD3–PE (BD). Propidium iodide was used to gate out dead cells. Results were acquired using an LSR I flow cytometer (BD) and analyzed in FlowJo software (Tree Star).
For BrdU incorporation analysis, CH12F3 cells (2 × 105/ml) were incubated for 30 min in culture medium containing 10 µM BrdU. Then, cells were harvested, washed twice with PBS, and fixed in cold 70% ethanol ON at 4°C. After removal of ethanol, DNA was denatured with 2 N HCl/0.5% Triton X-100 for 30 min at room temperature, neutralized with two washes of 0.1 M sodium tetraborate, pH 9, and resuspended in 70% ethanol. Cells were recovered by centrifugation, washed once with PBS, resuspended in 100 µl of blocking buffer (0.5% Tween 20 and 1% BSA in PBS) containing 10 µl mouse anti-BrdU antibody (BD), and incubated at room temperature for 30 min. After a wash with PBS, cells were incubated for 15 min at room temperature with goat anti–mouse Alexa Fluor 647 antibody diluted in blocking buffer. Finally, cells were washed with PBS once, resuspended in PBS containing 5 µg/ml propidium iodide, and analyzed using an Accuri flow cytometer.
For BrdU incorporation analysis, CH12F3 cells (2 × 105/ml) were incubated for 30 min in culture medium containing 10 µM BrdU. Then, cells were harvested, washed twice with PBS, and fixed in cold 70% ethanol ON at 4°C. After removal of ethanol, DNA was denatured with 2 N HCl/0.5% Triton X-100 for 30 min at room temperature, neutralized with two washes of 0.1 M sodium tetraborate, pH 9, and resuspended in 70% ethanol. Cells were recovered by centrifugation, washed once with PBS, resuspended in 100 µl of blocking buffer (0.5% Tween 20 and 1% BSA in PBS) containing 10 µl mouse anti-BrdU antibody (BD), and incubated at room temperature for 30 min. After a wash with PBS, cells were incubated for 15 min at room temperature with goat anti–mouse Alexa Fluor 647 antibody diluted in blocking buffer. Finally, cells were washed with PBS once, resuspended in PBS containing 5 µg/ml propidium iodide, and analyzed using an Accuri flow cytometer.
9
Peptide-based CD8+ T cell assay
10
Flow Cytometric Analysis of Intestinal Immune Cells
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