The commercially available antibodies to the following antigens were used: (i) from eBioscience, CD3 fluorescein isothiocyanate (FITC), CD8 phycoerytherin (PE) and cyanin 5.5 PE (Cy5.5PE), CD14 Cy7PE, CD16 Cy7PE, CD5 allophycocyanin (APC), CD19 Cy7PE and APC, CD20 FITC, CD45 PE, CD10 Cy7PE, CD34 Cy7PE, and tumor necrosis factor–α Alexa Fluor 700; (ii) from BioLegend, CD4 Brilliant Violet 605 (BV605) and BV785, CD8 Cy5PE, CD45RO BV570, and IFN-γ BV570; (iii) from Becton Dickinson, CD14 V500, CD3 BV605, IL-2 CF594PE, CD107a Cy5PE, granulocyte-macrophage colony-stimulating factor BV421, and macrophage inflammatory protein (MIP)–1β PE; (iv) from Beckman Coulter, CD27 Cy7PE; (v) from R&D systems, MIP-1α FITC. Alexa Fluor 647–conjugated mAb to detect the CAR19 molecule was described (32 (link)). All antibodies used in this study were titrated before use on the respective flow cytometer (see below). Routine assessment of CTL019 expansion and persistence and of CLL cells with a four-color, six-parameter Accuri C6 flow cytometer was performed as described previously (8 ).
Cd20 fitc
Manufactured by BioLegend
CD20-FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD20 antigen expressed on the surface of B cells. It is used for the identification and enumeration of B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd19 fitc, by BioLegend (2 mentions)
Cd16 fitc, by BioLegend (2 mentions)
Cd3 bv605, by BD (1 mentions)
Cd45 pe, by BioLegend (1 mentions)
Alexa fluor 700, by BioLegend (1 mentions)
Cd3 fluorescein isothiocyanate fitc, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Mip 1β pe, by Beckman Coulter (1 mentions)
Attune nxt, by Thermo Fisher Scientific (1 mentions)
Bv510 streptavidin, by BioLegend (1 mentions)
Streptavidin pe, by BioLegend (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Cd3 apc, by BioLegend (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
Cd27 apc, by Thermo Fisher Scientific (1 mentions)
Cd38 pe, by Thermo Fisher Scientific (1 mentions)
Fc receptor fcr block, by Miltenyi Biotec (1 mentions)
Fcr block, by Miltenyi Biotec (1 mentions)
Cd3 bv650, by BioLegend (1 mentions)
Cd34 apc, by BioLegend (1 mentions)
5 protocols using cd20 fitc
1
Multiparametric Flow Cytometry Analysis of CTL019 Cells
2
SARS-CoV-2 Spike Protein-Specific B Cell Detection
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To detect antigen-specific B cells, approximately 5 × 105 PBMCs were stained with 100 ng full-length biotinylated spike protein (Sino Biological) preincubated with Streptavidin-BV510 (Biolegend) at a 2:1 ratio for 1 hour at 4°C to ensure maximum staining quality before surface staining with CD20-FITC (Biolegend, 2H7) for an additional 30 minutes. Streptavidin PE (Biolegend) was used as a decoy probe to gate out SARS-CoV-2 nonspecific streptavidin binding. Samples were washed twice and resuspended in 200 μL FACS buffer before being analyzed on Attune NxT (Life Technologies).
3
Comprehensive B- and T-Cell Profiling Protocol
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For B-cell flow cytometry, 5 × 106 PBMCs were blocked with Fc receptor (FcR) Block (Miltenyi Biotec) and incubated in the dark on ice for 20 minutes with the following antibodies: IgD Brilliant Violet 421 (BioLegend 11-26c.2a), CD19 FITC (BioLegend HIB19), CD38 PE (eBioscience 90), and CD27 APC (eBioscience O323). B-cell subsets were gated out of CD19+ and defined as naive (N) CD19+IgD+CD27−; unswitched memory CD19+IgD+CD27+; double-negative CD19+IgD-CD27−; switched memory CD19+IgD-CD27+ CD38−; plasmablast (PB) CD19+IgD-CD27hiCD38hi CD138−; and plasma cells CD19+IgD-CD27hiCD38hi CD138+.
For T-cell flow cytometry, 5 × 106 PBMCs were blocked with FcR Block (Miltenyi Biotec) and incubated in the dark on ice for 20 minutes with the following antibodies: CD20 FITC (BioLegend 2H7), CD19 PC5.5 (BioLegend HIB19), CD3 APC (BioLegend), CD4 PE (BioLegend), and CD8 A750 (BioLegend). T-cell subsets were CD3+CD19− and sorted as CD3+CD4+CD8− or CD3+CD4−CD8+ T cells into RLT buffer (Qiagen) with 1% beta-mercaptoethanol and stored at −80°C for eventual RNA isolation. B- and T-cell flow analysis and sorting was performed on separate PBMC samples.
For T-cell flow cytometry, 5 × 106 PBMCs were blocked with FcR Block (Miltenyi Biotec) and incubated in the dark on ice for 20 minutes with the following antibodies: CD20 FITC (BioLegend 2H7), CD19 PC5.5 (BioLegend HIB19), CD3 APC (BioLegend), CD4 PE (BioLegend), and CD8 A750 (BioLegend). T-cell subsets were CD3+CD19− and sorted as CD3+CD4+CD8− or CD3+CD4−CD8+ T cells into RLT buffer (Qiagen) with 1% beta-mercaptoethanol and stored at −80°C for eventual RNA isolation. B- and T-cell flow analysis and sorting was performed on separate PBMC samples.
4
Isolation and Characterization of Preleukemic Cells in Down Syndrome
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Viable frozen peripheral blood samples from 2 DS patients with DS-associated myeloid preleukemia were obtained from the biobank commission of the Princess Máxima Center for Pediatric Oncology. Mononuclear cells were stained with a cocktail of the following antibodies: CD3-BV650 (Biolegend, Clone UCHT1, 300467, 1:100), CD4-PerCP/Cy5.5 (Biolegend, Clone OKT4, 317427, 1:200), CD8-BV785 (Biolegend, Clone SK1, 344739, 1:100), CD19-BV421 (Biolegend Clone HCD14, 30224, 1:100) , CD14-AF700 (Biolegend, Clone HCD14, 325614, 1:100), CD56-BV711 (Biolegend, Clone HCD56, 318335, 1:50), CD34-APC (Biolegend, Clone 561, 343607, 1:50), CD38-PE (Biolegend, Clone HIT2, 303505, 1:50) , CD33-PE/Cy7 (Biolegend, Clone WM53, 303433, 1:100), CD117-PE-dazzle594 (Biolegend, Clone 104D2, 1:100), CD16-FITC (Biolegend, Clone 3G8, 302005, 1:100), CD20-FITC (Biolegend, Clone IVB201, 302303, 1:100). Bulk T-cells (CD3+/CD4+ and CD3+/CD8+) and preleukemic blast cells were sorted with the Astrios-EQ. Preleukemic blast cells were sorted according to the diagnostics flow data. Cell pellets were used for DNA isolation. Public data was used for the other 4 myeloid preleukemia samples11 (link).
5
Multiparametric Immune Cell Profiling
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The following anti-human antibodies were used for the lineage the cocktail: CD3-FITC, CD4-FITC, CD8a-FITC, CD14-FITC, CD15-FITC, CD16-FITC, CD19-FITC, CD20-FITC, CD33-FITC, CD34-FITC, FcεRI-FITC, and CD203c-FITC (all from Biolegend). In addition, these anti-human antibodies were used: CD335 (NKp46)-PE/Cy7, CD294 (CRTH2)-PE, CD127-BV421 from Biolegend, CD56-APCeF780 from eBioscience, and CD117 (ckit)-APC from BD Bioscience. For intracellular transcription factor staining the following anti-human antibodies were used: Tbet-PE/CF594, GATA3-PE/Cy7, and RORγt-PE from BD Bioscience. Corresponding isotype control antibodies were used as controls.
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