Splenocytes or lymph node mononuclear cells (LMNCs) were resuspended in FACS staining buffer (PBS, 2% BSA, 5 mM EDTA, and 0.03% NaN3), stained, and then fixed in Cytofix/Cytoperm (BD Bioscience, San Jose, CA). T-cell activation panel consisted of CD3 AF700 (500-A2, BD Pharminogen, San Diego, CA), CD4 PE (GK1.5, BD Pharminogen, San Diego, CA, USA), CD8 PE-CF594 (53-6.7 BD Horizon, San Jose, CA, USA), and CD69 APC (H1.2F3, Biolegend, San Diego, CA, USA). Germinal center B-cell panel consisted of CD3 AF700 (500-A2, BD Pharminogen, San Diego, CA, USA), B220 PE-Cy7 (RA3-6B2 BD Pharminogen, San Diego, CA, USA), CD95 PE-CF594 (Jo2, BD Pharminogen, San Diego, CA, USA), and GL7 BV421 (GL7, BD Horizon, San Jose, CA, USA). Memory T-cell Panel consisted of CD3 AF700 (500-A2, BD Pharminogen, San Diego, CA, USA), CD4 BV650 (GK1.5, BD Horizon, San Jose, CA, USA), CD8 BV786 (53-6.7, BD Horizon, San Jose, CA, USA), CD62L APC (MEL-14 Biolegend, San Diego, CA, USA), and CD44 FITC (IM7, Biolegend, San Diego, CA, USA). Memory B-cell panel consisted of CD3 AF700 (500-A2, BD Pharminogen, San Diego, CA, USA), B220 PE-Cy7 (RA3-6B2, BD Pharminogen, San Diego, CA, USA), and CD73 AF650 (TY/23, BD Pharminogen, San Diego, CA, USA).
B220 pe cy7
Manufactured by BD
Sourced in United States
The B220-PE-Cy7 is a fluorochrome-conjugated antibody specific for the B220 antigen. It is designed for use in flow cytometry applications to identify and characterize B cells.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 pe, by Thermo Fisher Scientific (2 mentions)
Fvs 440uv, by BD (2 mentions)
Cd45 bv786, by BD (2 mentions)
Cd8 bv605, by BD (2 mentions)
Cd4 v500, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Cd19 percp cy5, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd38 fitc, by BD (2 mentions)
Streptavidin apc, by Thermo Fisher Scientific (2 mentions)
Gl7 pe, by BD (2 mentions)
Lsrfortessa x 20 instrument, by BD (2 mentions)
Cd3ε pacificblue, by BD (2 mentions)
Ethidium monoazide bromide ema, by Thermo Fisher Scientific (2 mentions)
Cd86 pe, by BioLegend (1 mentions)
Cd80 percp cy5, by BioLegend (1 mentions)
Mhcii pacificblue, by BioLegend (1 mentions)
Cd40 pe, by BioLegend (1 mentions)
Cd21 fitc, by BioLegend (1 mentions)
Cd8 fitc, by BioLegend (1 mentions)
15 protocols using b220 pe cy7
1
Multiparameter Flow Cytometry Panels
2
Multiparametric Flow Cytometry of Murine Immune Cells
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Murine spleens and lymph nodes were dissected and passed through 70 µm strainers. After pre‐incubation with Zombie™ dye and Fc‐block (BioLegend), cells were stained with antibodies against B220‐FITC, B220‐PE‐Cy7, CD19‐FITC, CD25‐APC, CD69‐PerCP‐Cy5.5, CD95‐PE (all BD Bioscience), CD4‐PE, CD8‐FITC, CD11b‐BV510, CD21‐FITC, CD23‐APC, CD40‐PE, CD80‐PerCP‐Cy5.5, CD86‐PE and MHC‐II‐PacificBlue (all BioLegend). T cells were stained for interferon (IFN)‐γ‐APC (BioLegend) and IL‐17A‐PE (BD Bioscience) after 4‐h incubation with 50 ng/mL PMA, 0.5 μg/mL ionomycin and 1 μL/mL Golgi‐Plug; regulatory T cells were determined by intracellular staining for Foxp3‐PE (BD Bioscience).
3
Analyzing Tumor-Specific Immune Responses
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To systematically investigate the in vivo antitumour immune responses against mimic distant tumours, the tumours were harvested and treated with the tissue dissociation kit (Miltenyi Biotec, Germany) to produce a single-cell suspension according to the specified procedures. The harvested cells were further stained with several fluorochrome-conjugated antibodies: CD45-FITC (BD, Catalog: 561088), CD3-PerCP-Cy5.5 (BD, Catalog: 551163), CD4-BV510 (BD, Catalog: 563106), CD8-BV421 (BD, Catalog: 563898), NKp46-APC (Biolegend, Catalog: 137608), B220-PE-Cy7 (BD, Catalog: 552772) and Foxp3-PE (eBioscience, Catalog: 12–4771) and then analysed by FCM. For the analysis of the memory T cells, spleen cells of mice were harvested and stained with anti-CD3-FITC (Biolegend, Catalog: 100306), anti-CD4-PE-Cy7 (eBioscience, Catalog: 25–0041), anti-CD8-PerCP-Cy5.5 (eBioscience, Catalog: 45–0081), anti-CD44-PE (eBioscience, Catalog: 12–0441) and anti-CD62L-APC (eBioscience, Catalog: 17–0621) antibodies and then analysed by FCM. All antibodies were diluted ~100 times.
4
Airway-Draining Lymph Node Analysis
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Airway‐draining lymph node cells (posterior mediastinal, tracheobronchial, parathymic, ADLN) were removed from mice and physically disaggregated. Staining of surface antigens was performed as described previously (Gorman et al. 2007 ) using anti‐rat monoclonal antibodies supplied by BD Biosciences (CD86‐BIO, streptavidin‐PE‐Cy5, B220‐PE‐Cy7, MHC class II‐FITC). At least 10,000 cells of interest were collected using the FACS LSRII (BD Biosciences) flow cytometer. Data were analyzed using FlowJo software (v9.5.2, TreeStar Inc., Ashland OR).
5
Splenocyte Isolation and Immunophenotyping
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Concentration of splenocyte suspension was adjusted to 5 × 105 cells/100 μl with PBS and stained with Fixable Viability Stain (FVS)-440UV (Cat. 566332, BD Biosciences, Oxford, United Kingdom) at 1 : 1000 ratio for 15 min in the dark. Staining was terminated by washing with the same volume of BSA staining buffer (Cat. 554657, BD) twice before surface antigens staining. After preincubation with Fc block (Cat. 553141, BD) at 1 : 50 ratio for 5 min, antibodies from CD45-BV786 (Clone 30-F11, BD), CD3-PE-Cyanine5 (Clone 145-2C11, BD), B220-PE-Cy7 (Clone RA3-6B2, BD), CD4-FITC (Clone RM4-5, BD), and CD8-BV605 (Clone 53-6.7, BD) were added at 1 : 50 ratio and incubated in the dark for 15 min at 4°C. Cells were then washed twice and resuspended in 0.5 ml cold PBS. Cell analysis was performed using a flow cytometry (CytoFLEX LX, Beckman Coulter, CA, United States) and CytExpert analysis software version 2.3 (Beckman Coulter) following manufacturer's instructions.
6
Activated Tfh Cells and Plasma Cells Profiling
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Activated Tfh cells in the spleen were labeled with FVS-440UV, CD45-BV786, CD3-FITC (Clone 145-2C11, BD), CD4-V500, CD8-BV605, and CXCR5-PE (Clone 2G8, BD) following surface antigen staining procedure. Subsequently, cell pallets were suspended in 1 ml fixation buffer (Cat. 554655, BD) for 15 min under room temperature and washed with 1 ml perm/wash buffer (Cat. 557885, BD) twice. Anti-mouse Bcl-6-Alexa Fluor 647 (Clone K112-91, BD) was added at 1 : 50 ratio and incubated in the dark for 30 min at 4°C. Cells were then washed twice and resuspended in cold PBS for flow cytometry assay. Plasma cells in peripheral blood cells (PBMCs) were labeled with FVS-440UV, CD45-BV786, CD3-FITC, B220-PE-Cy7, CD19-APC-cy7 (Clone 1D3, BD), and CD138-BV421 (Clone 281-2, BD) following the procedure described earlier.
7
Quantitative Splenocyte Profiling
8
Comprehensive B Cell Immunophenotyping
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Bone marrow of Myce and littermate wild‐type control were harvested into single‐cell suspension with red cell lysis and stained with CD19‐Pacific Blue (1D3, BD, NJ, USA), B220‐PE cy7 (RA3‐6B2, BD), CD24‐APC (M1/69, in house), IgM‐FITC (331.12, in house), CD43‐PE (S7, BD). Spleen red cell lysed single cell suspensions were stained with CD19‐BB700 (1D3, BD), B220‐APC (Thermo Fisher, Waltham, USA), CD23‐PE cy7 (B3B4, Thermo Fisher), CD21‐BV421 (7E9, BioLegend, San Diego, USA), CD138‐PE (281–2, BD). The peritoneal cavity wash was stained with CD19‐PE (ID2, in house), B220‐APC (RA3‐6B2, Thermo Fisher), CD23‐PE cy7 (B3B4, Thermo Fisher), CD5‐BB700 (53–7.3, BD), CD11b‐eFlour450 (M1/70, Thermo Fisher). Lymph node and thymus of single cell suspensions were stained with CD19‐BUV395 (1D3, BD), (TCRβ‐PE (H57‐597, BD), CD4‐BV421 (GK1.5, BioLegend) and CD8a‐PerCp/Cy5.5 (53–6.7, eBioscience, San Diego, USA). Flow cytometry analysis was performed on a BD LSRFortessa X‐20 analyzer (BD).
9
Multiparameter Flow Cytometry Analysis
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For cell surface staining, empirically determined dilutions of primary mAbs were used to stain single cell suspensions on ice for 20 min in FACS buffer (PBS with 2% FBS, 0.1% NaN3, and 1 µM EDTA). All mAbs were purchased from BioLegend unless otherwise indicated. The following mAb clones were used: B220-PE-Cy7, CD4-PE-Cy7, CD8α-PerCP-Cy5.5, CD11b-PE-Cy7, CD11c-FITC, CD25–Alexa Fluor 647, CD44-FITC, CD45.1-FITC, CD45.2-BV605, CD62L-PE-Cy7, CD103-biotin (followed by streptavidin-BV711; BD), Foxp3-PE (eBioscience), Helios-APC, I-A(b)-PE, Ki-67–Alexa Fluor 647, and TCR-β–Pacific blue. Dead cells were excluded using Fixable Viability Dye eFluor780 (eBioscience). Intracellular staining to detect Foxp3 expression was performed according to the manufacturer’s instructions, using the manufacturer’s protocol for Foxp3 staining in 96-well plates (eBioscience). Cells were analyzed using an LSR-II flow cytometer (BD) equipped with 405-, 488-, 552-, and 640-nm lasers. Flow cytometry data were processed using FlowJo version 9.7.5 software (Tree Star).
10
Flow Cytometric Analysis of Lymphocytes
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