Splenocytes from Balb/c mice were isolated and erythrocytes were lysed by treatment with FACS lysing solution (BD Biosciences, USA), prepared according to manufacturer’s instructions. Cells were washed in PBS and suspended in 1% (w/v) bovine serum albumin (Sigma) prepared in PBS. Approximately 106 splenocytes were pelleted and stained for 30 min at 4°C in the dark with 20 μL of fluorescent monoclonal antibodies against: CD3-PE, CD4-FITC or CD4-Alexa Fluor 647, CD8-PerCP, B220-APC and CD45RB-FITC (BD Biosciences), previously titrated and mixed. Splenocytes were washed twice, suspended in 300 μL of PBS and read in a BD Accuri C6 Flow Cytometer (BD, USA). Cell populations were analyzed offline using FlowJo software (Tree Star, USA). For the in vivo cytotoxicity analysis, CFSE-stained cells were readily analyzed without additional markers.
B220 apc
Manufactured by BD
Sourced in United States
The B220-APC is a fluorescent-labeled antibody that binds to the B220 cell surface antigen, which is expressed on mature B cells and a subset of T cells. It is used in flow cytometry applications to identify and quantify B lymphocyte populations.
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Lab products found in correlation
Cd4 pe, by BD (5 mentions)
Cd3 fitc, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Cd8 percp, by BD (2 mentions)
Cd3 pe, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Cd11c fitc, by BD (2 mentions)
Cd4 apc, by BD (2 mentions)
Ly6c pe, by BD (2 mentions)
Cd8 pe cy7, by BD (2 mentions)
Cd69 fitc, by BD (2 mentions)
Cd86 pe, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Cd43 fitc, by BD (2 mentions)
Igm fitc, by BD (2 mentions)
Foxp3 pe, by BD (2 mentions)
Cd11c pe, by BD (2 mentions)
Gr1 pe, by BD (2 mentions)
Bp1 pe, by BD (2 mentions)
Gr1 fitc, by BD (2 mentions)
19 protocols using b220 apc
1
Murine Splenocyte Isolation and Flow Cytometry
2
Somatic Hypermutation Analysis Workflow
3
Immunophenotyping of Murine Macrophages
4
Internalization Kinetics of IgM Receptor
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Single cell suspensions of splenocytes were prepared with 1x PBS, and red blood cells were lysed using 1x BD Pharm Lyse buffer (BD Biosciences).
Internalization of IgM-FITC. After the cells were washed, they were stained on ice with anti-IgM-FITC for 15 minutes and washed with ice-cold PBS. The cells were resuspended in 37°C pre-warmed 1x PBS/3% FCS and incubated at 37°C. At the time points 0, 5, 10, 15, 20, 25, and 30 minutes, an aliquot was stopped with ice-cold PBS. The cells were additionally stained with B220-APC (BD Pharmingen) and eFluor450 (eBioscience) on ice for 15 minutes and washed again with ice-cold PBS.
Internalization of IgM-pHrodo. In total, 100 μg goat anti-mouse IgM (clone 1020-01) was labeled using a pHrodo Red Microscale Labeling Kit (Molecular Probes by Life Technologies) according to the manufacturer's instructions. Then, 1 × 106 cells were resuspended in 3 ml PBS/30 μl IgM-pHrodo and incubated at 37°C. At the time points 0, 15, 30, 45, 60, 75, and 90 minutes, 300 μl was stopped with ice-cold PBS, centrifuged and stained with B220-APC and eFluor450.
Internalization of IgM-FITC. After the cells were washed, they were stained on ice with anti-IgM-FITC for 15 minutes and washed with ice-cold PBS. The cells were resuspended in 37°C pre-warmed 1x PBS/3% FCS and incubated at 37°C. At the time points 0, 5, 10, 15, 20, 25, and 30 minutes, an aliquot was stopped with ice-cold PBS. The cells were additionally stained with B220-APC (BD Pharmingen) and eFluor450 (eBioscience) on ice for 15 minutes and washed again with ice-cold PBS.
Internalization of IgM-pHrodo. In total, 100 μg goat anti-mouse IgM (clone 1020-01) was labeled using a pHrodo Red Microscale Labeling Kit (Molecular Probes by Life Technologies) according to the manufacturer's instructions. Then, 1 × 106 cells were resuspended in 3 ml PBS/30 μl IgM-pHrodo and incubated at 37°C. At the time points 0, 15, 30, 45, 60, 75, and 90 minutes, 300 μl was stopped with ice-cold PBS, centrifuged and stained with B220-APC and eFluor450.
5
Immune Cell Profiling by Flow Cytometry
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Cells recovered from BAL samples were stained with antibodies to CD4-PE, B220-APC, CD8-PerCP, CD11b-FITC, CD11c-APC, Ly6C-PerCP (BD Biosciences or BioLegend) and analyzed by flow cytometry on a FACS Canto (BD Biosciences), using FlowJo software (Tree Star). Different combinations of antibodies were used as indicated in the text.
6
Immunophenotypic Analysis of Splenocytes
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Immunophenotypic analyses of splenoctyes from animals were assessed by flow cytometry. Antibodies to stain for MDSC were CD11b-APC (Clone M1/70; BD Biosciences), Ly6G-FITC (Clone 1A8; BD Biosciences), Ly6C-PE (Clone AL-21; BD Biosciences); for dendritic cells were CD11c (Clone HL3; BD Biosciences); for B cells B220-APC (Clone RA3–6B2; BD Biosciences), CD3-FITC (Clone 145–1011; BD Biosciences). T regulatory cells with a phenotype of CD4+CD25+FoxP3+ were evaluated using a commercially available kit (eBiosciences, San Diego, CA). For T cell activation markers, cells were stained with antibodies specific for CD4-PE-Cy7 (Clone RM4–5; BD Biosciences), CD8-PE-Cy7 (Clone 53–6.7; BD Biosciences), CD62L-PE (Clone MEL-14; BD Biosciences), and CD44-Bv650 (Clone IM7; Biolegend). To determine Th1 and Th2 phenotypes, cells were stained using fluorochrome conjugated antibodies targeted CXCR3-PE-Cy7 (Clone CXCR13–173; Biolegend), CCR4-PE (Clone 2G12; Biolegend), and CCR6-APC (Clone CK4-L3; BD Biosciences). Cells were incubated on ice for 30 minutes, washed, and fixed in PBS containing 1% formalin for flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).
7
Comprehensive Lymphocyte Phenotyping by Flow Cytometry
8
Multicolor Flow Cytometry of AGM Cells
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AGM cells were stained with anti-CD31PE-Cy7 (eBioscience, 12-0311-82) and cKit APC (Becton Dickinson, 553356) antibodies and adult hematopoietic cells with anti-CD31PE (BD, 561073), Ly6cAPC-Cy7 (BD, 560596), CD4PE (BD, 557308), CD8PE (BD, 553032), and B220APC (BD, 553092) antibodies. Wild-type AGM cells were used as negative control for the BRE GFP AGM cells. Unstained and single marker stained cells were used to define the gates for fluorescence-activated cell sorting (FACS) analysis and cell sorting. Cells were analyzed on a FACSAria SORP or FACSAria III (BD) with FloJo software. Dead cell exclusion was with Hoechst 33258 (Molecular Probes).
9
Flow Cytometry Analysis of Eμ-Myc Lymphoma
10
Multicolor Flow Cytometry for T Cell Analysis
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