For Treg cells, isolated PBMCs were stained with anti-CD4-FITC (RPA-T4/555346), CD25-APC (M-A251/555434), and CD45RA-PerCP-Cy 5.5 (HI100/563429) antibodies (BD Biosciences, San Jose, CA, USA) for 45 min, and antibody stained samples were washed twice. After intracellular staining, Treg cell frequencies were analyzed by a BD FACSVerse (BD Biosciences) flow cytometer. For MDSCs, isolated PBMCs were stained with anti-CD3-BV421 (UCHT1/562426), CD19-BV421 (HIB19/562440), CD56-BV421 (NCAM16.2/562751), CD20-BV421 (2H7/562873), CD11b-BB515 (ICRF44/564517), CD15-PerCP-Cy 5.5 (HI98/560828), CD14-APC (M5E2/555399), and HLA-DR-PE (G46-6/555812) antibodies (BD Biosciences) for 45 min, washed twice, and analyzed by a BD FACSVerse (BD Biosciences) flow cytometer. For 7-AAD and propidium iodide staining, isolated PBMCs were stained with 7-AAD (Biolegend, San Diego, CA, USA) or PI (BD Biosciences) for 10 min and then analyzed on a BD FACSVerse (BD Biosciences). Gating strategies are shown in Supplementary Fig. S1 . PBMC viability before MDSC analysis is shown in Supplementary Fig. S2 .
Cd20 bv421
Manufactured by BD
Sourced in United States
CD20-BV421 is a fluorochrome-conjugated monoclonal antibody that binds to the CD20 antigen expressed on the surface of B lymphocytes. It can be used for the identification and enumeration of B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 bv421, by BD (2 mentions)
Cd56 bv421, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Cd11b bb515, by BD (2 mentions)
Cd19 bv421, by BD (2 mentions)
Hla dr pe g46 6 555812, by BD (2 mentions)
Anti cd4 fitc, by BD (1 mentions)
Cd14 apc, by BD (1 mentions)
7 aad, by BioLegend (1 mentions)
7 aminoactinomycin d 7 aad, by BioLegend (1 mentions)
Cd15 percp cy5, by BD (1 mentions)
Foxp3 pe 259d c7 560046, by BD (1 mentions)
Cd45ra percp cy5, by BD (1 mentions)
Cd3 bv786, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cd31 alexa 647, by BD (1 mentions)
Cd138 pe, by Thermo Fisher Scientific (1 mentions)
Cd49d apc, by BD (1 mentions)
Fcr blocking, by BD (1 mentions)
3 protocols using cd20 bv421
1
Multicolor Cytometry for Treg and MDSC
2
Multiparametric Flow Cytometry Phenotyping
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For MDSCs, isolated PBMCs were stained with anti‐CD3‐BV421 (UCHT1/562426), CD19‐BV421 (HIB19/562440), CD56‐BV421 (NCAM16.2/562751), CD20‐BV421 (2H7/562873), CD11b‐BB515 (ICRF44/564517), CD15‐PerCP‐Cy 5.5 (HI98/560828), CD14‐APC (M5E2/555399), and HLA‐DR‐PE (G46‐6/555812) antibodies (BD Biosciences, San Jose, CA, USA) for 45 min. For lectin‐type oxidized LDL receptor 1 (Lox‐1) expression on PMN‐MDSCs, lox‐1‐BV421 (15C4/358610) (Biolegend, San Diego, CA, USA) was used. For Treg cells, isolated PBMCs were stained with anti‐CD4‐FITC (RPA‐T4/555346), CD25‐APC (M‐A251/555434), CD45RA‐PerCP‐Cy 5.5 (HI100/563429), and Foxp3‐PE (259D/C7/560046) antibodies (BD Biosciences) for 45 min. For CD8+ T cells, isolated PBMCs were stained with anti‐CD8‐APC (RPA‐T8/555369) (BD Biosciences), CD39‐PE‐Cy7 (A1/328212) (Biolegend), and PD‐1‐PerCP‐Cy5.5 (EH12.1 /561273) (BD Biosciences) antibodies. Samples were washed twice and then read on a BD FACSVerse (BD Biosciences) flow cytometer. Dead cells were excluded using 7‐Amino‐Actinomycin D (7AAD) (Biolegend). Gating strategies for PMN‐MDSCs, M‐MDSCs, and CD8+ T cells are shown in Supporting information Fig. 1A‐C. All the process of T‐cell assays and flow cytometry analysis (linear axis) adhered to the guidelines of MIATA compliant and Cossarizza [30 ].
3
Phenotyping of Rhesus Macaque PBMCs
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Frozen PBMCs were thawed, and cell viability was consistently >90%. After the incubation with the FcR blocking (BD) and Aqua Dead Cell Stain (Molecular Probes), as described in Section “Flow Cytometry of BM PCs ,” the cells were washed with FACS buffer and surface-stained for 15 min at 4°C with antibodies against CD3 BV786 (BD), CD20 BV421 (BD), CD138 PE (eBioscience), CD80 clone L307.4 APC-H7 (BD), HLA-DR clone L243 PerCP-Cy5.5 (BD), CD14 clone M5E2 BV786 (BD), CD16 clone 3G8 BV786 (BD), CD98 FITC (eBioscience), and CD49d APC (BD) or CD31 Alexa 647 (BD). Cells were washed twice with FACS buffer and incubated in 100 μl of Cytofix/Cytoperm (BD) for 20 min at 4°C. Cells were then washed twice in Perm/Wash buffer (BD) and intracellularly stained in 100 μl of Perm/Wash buffer for 15 min at 4°C with IgG clone G18-145 PE-Cy7 (BD), followed by two washes in Perm/Wash buffer and two in FACS buffer. All Abs were previously titrated for optimal staining of rhesus macaque PBMCs. Samples were collected on a FACS LSRII (BD Immunocytometry Systems) and analyzed using FlowJo software.
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