Immunophenotyping of lymphocytes was performed by multicolor flow cytometry. Heparinized whole blood samples were immediately stained for 15 minutes with the following antibodies: CD3-APC/Cy7 (UCHT, BioLegend; San Diego, CA, USA), CD4-HorizonV500 (RPA-T4, BD Biosciences; San Jose, CA, USA), CD45RA-BV421 (HI-100, BioLegend), CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), CXCR3-APC (1C6, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PE/Cy7 (G043H7, BioLegend), PD-1-FITC (EH12.2H7, BioLegend), CD19-BV421 (HIB19, BioLegend), CD20-APC (2H7, BioLegend), CD27-APC/Cy7 (O323, BioLegend), and CD38-FITC (HIT2, BioLegend). Circulating Tfh cells were defined as CD3+CD4+CD45RA-CXCR5+ cells, and Tfh1, Tfh2, or Tfh17 cell subsets as CXCR3+CCR6- cells, CXCR3-CCR6- cells, or CXCR3-CCR6+ cells among Tfh cells, respectively [19 (link), 20 (link)]. Activated Tfh cells were defined as the CCR7lowPD-1high cells among Tfh cells [26 (link)]. CD19+CD20-CD27+CD38+ cells were defined as plasmablasts. Red blood cells were lysed with FACS Lysing Solution (BD Biosciences). All samples were analyzed with a FACS Aria III (BD Biosciences), and data were analyzed with FlowJo v.7.6.4 Software (Tree Star, Stanford University, CA, USA). Proportions of lymphocyte subsets were determined by the combination of surface marker staining, with exclusion of doublets by forward and side scatter.
Cd20 apc
Manufactured by BioLegend
Sourced in United States
CD20-APC is a fluorescently labeled antibody that binds to the CD20 antigen, which is expressed on the surface of B cells. The APC (Allophycocyanin) fluorescent dye is conjugated to the antibody, allowing for the detection and analysis of CD20-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (1 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Ccr7 pe cy7, by BioLegend (1 mentions)
Facs lysing solution, by BD (1 mentions)
Cd38 fitc, by BioLegend (1 mentions)
Cd19 bv421, by BioLegend (1 mentions)
Cd27 apc cy7, by BioLegend (1 mentions)
Ccr6 pe 11a9, by BD (1 mentions)
Pd 1 fitc eh12.2h7, by BioLegend (1 mentions)
Fura red, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Cd3 apc, by BD (1 mentions)
Cd56 fitc, by BD (1 mentions)
Cd3 apc h7, by BD (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
Cd20 apc, by BD (1 mentions)
Cd19 percp cy5, by BD (1 mentions)
Cd8 percp cy5, by BD (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
3 protocols using cd20 apc
1
Multicolor Flow Cytometry to Characterize Tfh Cells
2
PBMC Calcium Flux Assay with Fura Red and Fluo-4
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Example 7
PBMC Staining with 3 μM Fura Red and Fluo-4
Fura Red and FLUO-4 (each from Invitrogen, Life Technologies Corp., Carlsbad, Calif.) calcium stain was added to serum free RPMI medium to a final concentration of 3 μM for each dye, then incubated at 37° C. for 30 minutes. PBMCs mixed 1:1 with media containing 10% FCS and incubated an additional 10 minutes. The cells were washed 2× with media containing 10% FCS. Cells were resuspended in RPMI with 10% human serum.
The resuspended cells were incubated with or without the addition of 100 μg/mL IMPRIME PGG at 37° C. for one hour. The monoclonal antibody CD20 APC (BioLegend, Inc., San Diego, Calif.) was added to each tube for 10 minute incubation before washing 2× with RPMI containing 10% FCS. Cells were resuspended in RPMI with 10% FCS for FACS analysis. Calcium flux was induced by stimulating the B cells with goat anti-human IgM at 2.6 μg/mL. Results are shown in
3
Flow Cytometry Analysis of Blood Cells
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Absolute numbers of thrombocytes and ferritin levels were routinely measured according to the established diagnostic guidelines. Absolute numbers of lymphocytes were determined with Multitest 6-color reagents (BD Biosciences, San Jose, Calif) according to the manufacturer's instructions. For additional flow cytometry, PBMCs were resuspended in PBS containing 0.5% (wt/vol) BSA and 0.01% sodium azide and then incubated with saturating concentrations of fluorescently labeled conjugated mAb antibodies. Patient samples were analyzed simultaneously with PBMCs from healthy donors. The following directly conjugated mAbs were used: CD3-APC, CD3-APC-H7, CD4-PE-Cy7, CD4-PerCP-Cy5.5, CD8-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD20-APC, and CD56-FITC (from BD Biosciences); IgD-PE and gamma-1 isotype (from BD Pharmingen, San Diego, Calif); CD20-APC (from Biolegend San Diego, Calif); CD16-FITC and CD27-FITC (from Sanquin, Amsterdam, The Netherlands); and CD45RA-PE (RD-1) (from Beckman Coulter, Brea, Calif). Analysis of cells was performed using a FACS Calibur or Canto-II flow cytometer (from BD Biosciences) and FlowJo software.
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