Samples were processed into single cell suspensions and stained in FACS buffer (PBS + 2% FBS + 0.1% sodium Azide) with antibody for 45 minutes on ice followed by secondary (APC- or PE-conjugated streptavidin) binding for 20 minutes. Antibodies used were CD8-BUV395 (RPA-T8, BD Biosciences), CD4-Bv605 (RPA-T4, BD Biosciences), CD3-APC (SP34–2, BD Biosciences), FGFR4-PE (4FR6D3, BioLegend), CD19-PE (HIB19, Biolegend), PDL1-APC (MIH1, BD Bioscience), and PDL2-Bv421 (MIH18, BD Biosciences). Surface CAR was detected using biotinylated CD19 with Fc-tag (Acro Bio) or biotinylated FGFR4-Fc (Sino Bio). For evaluation of FGFR4 binders identified from phage screening, 500 nmol/L VH or Fab proteins were incubated with cells at 4°C for 30 minutes followed by PE-conjugated anti-human Fc antibody (Miltenyi Biotec, 130–101–576) for 30 minutes at 4°C. Cells were then analyzed by flow cytometry using BD LSR II. Tumor cell surface antigen was quantified using Quanti-Brite beads (BD Biosciences) according to the manufacturer's instructions. Quantibrite beads and corresponding tumor cells stained for FGFR4 antigen were acquired on a BD LSRII and analyzed using GraphPad Prism. Data were analyzed by FlowJo V10.
Cd19 pe hib19
Manufactured by BioLegend
CD19-PE (HIB19) is a fluorescent-labeled antibody used for the detection and quantification of CD19-positive cells in flow cytometry applications. CD19 is a cell surface protein expressed on B lymphocytes, and the PE (phycoerythrin) fluorescent label allows for the visualization of CD19-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Quantibrite beads, by BD (2 mentions)
Cd8 buv395 rpa t8, by BD (2 mentions)
Cd4 bv605 rpa t4, by BD (2 mentions)
Cd3 apc sp34 2, by BD (2 mentions)
Fgfr4 pe 4fr6d3, by BioLegend (2 mentions)
Pdl1 apc mih1, by BD (2 mentions)
Pdl2 bv421 mih18, by BD (2 mentions)
Pe conjugated anti human fc antibody, by Miltenyi Biotec (2 mentions)
Lsr 2, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Nkp46 pe cy7 9e2, by BioLegend (1 mentions)
Cd4 apc rpa t4, by BioLegend (1 mentions)
Fc block, by BD (1 mentions)
Perm buffer 3, by BD (1 mentions)
Cxcr4 pe, by BD (1 mentions)
Cd11b apc, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Cytofix buffer, by BD (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd19 pe hib19
1
Quantifying Cell Surface Antigens by Flow Cytometry
2
Isolation and Purification of Immune Cells
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Cryopreserved PBMC from patients and healthy controls were thawed and stained with fluorochrome-conjugated Ab against: CD3 (FITC; UCHT1), CD4 (BV421; OKT4), CD8 (APC; HIT8), CD19 (PE; HIB19), and NKp46 (PE-Cy7; 9E2) all from BioLegend. Hereafter, the PBMC were subjected to a 4-way fluorescence-activated cell sorting (FACS) into CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD3- CD19+ B cells, and CD3- NKp46+ NK cells using a FACS Aria II (BD Biosciences, CA, USA). After FACS the cells were immediately lysed in Qiazol lysis buffer and frozen at −80 °C. The purity of isolated cells was 99.3% for CD4+ T cells, 99.5% for CD8+ T cells, 99.6% for B cells, and 98.1% for NK cells.
3
Quantification of Cell Surface Antigens
4
Flow Cytometric Analysis of BLaER1 Transdifferentiation
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For flow cytometric analysis of BLaER1 transdifferentiation and surface marker expression, 1 × 106 native or transdifferentiated BLaER1 cells were collected, washed once in FACS buffer (10% FCS, 0.1% Sodium acetate in PBS; 10 min, 300g, 4°C) and stained with CD11b-APC (M1/70, Biolegend), CD19-PE (HIB19, Biolegend), CD14-PacBlue (M5E2, Biolegend), CD163-PE (GHI/61, BD), CD206-APC (19.2, BD), CD11c-VioBlue (MJ4–27G12, Miltenyi), CD4-APC (RPA-T4, Biolegend), CXCR4-PE (12G5, BD), CCR5-PE (T21/8, Biolegend) or respective isotype controls (Biolegend, BD, Miltenyi) and Fixable Viability Dye eFluor 780 (Thermo Fischer) in presence of FC Block (BD, 20 min, 4°C). Stained cells were washed in FACS buffer twice and fixed in 2% paraformaldehyde (30 min, RT), before analyzing on a LSR II instrument (BD). For readout of HIV-1-mCherry infection, up to six wells of a 96-well plate were pooled and stained with CD11b-APC and Fixable Viability Dye eFluor 780 as detailed above. For intracellular SAMHD1 staining, cells were fixed in Cytofix Buffer (BD; 37°C, 10 min), subsequent to cell surface staining, and permeabilisation with Perm Buffer III (BD; 2min on ice), before staining with anti-SAMHD1 (12586–1-AP, Proteintech) or an isotype control (CST; 60 min, RT) and anti-rabbit IgG-DyLight 405 (Thermo Fischer; 60 min RT). Infected cells were analyzed on a BD LSRFortessa.
5
Multiparameter Flow Cytometry Analysis of PBMCs
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Flow cytometry was performed on PBMCs from both the influenza vaccine immunized volunteers and those from the VRC 902 Study. There were 2 panels of antibodies; one for CXCR5+CD4+ T cell subsets, and the second panel for memory B cells. For the staining of T cells, the following antibodies were used: CXCR5-AF488 (RF8B2, BD Biosciences, San Diego CA), PD1-PE (eBioJ105, eBioscience, San Diego, CA), CD45RO-PE/CF594 (UCHL1, BD Biosciences), CCR6-PerCP/Cy5.5 (G034E3, Biolegend, San Diego, CA), ICOS-PE/Cy7 (C398.4A, Biolegend), CCR7-AF647 (3D12, BD Biosciences), CXCR3-AF700 (1C6/CXCR3, BD Biosciences), CD4-APC/eFluor 780 (SK3, eBioscience), CD27-V450 (M-T271, BD Biosciences), CD8-BV510 (SK1, BD Biosciences), and CD3-BV605 (SK7, BD Biosciences). For B cells, IgD-AF488 (IA6-2, Biolegend), CD19-PE (HIB19, Biolegend), CD38-PE/Cy7 (HB-7, Biolegend), and CD27-BV450 (M-T271, BD Biosciences) were used. To exclude dead cells, Zombie UV Fixable Viability Kit (Biolegend) was used. For compensation, UltraComp eBeads (eBioscience) were used on each day the experiment was performed.
6
Multiparametric Flow Cytometry Profiling of CLL Cells
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After treatment with selinexor, leptomycin B or brefeldin A (BFA, GolgiPlug, BD Bioscience) for the required timepoint, Fc receptors were blocked on CLL cells with 10% human serum for 15 min then cells were stained with CD19-PE (HIB19, Biolegend) or CD19-PB, CD5-PerCP (UCHT2, Biolegend) or CD5-APC, ULBP-1-PE (170818, R&D Systems), ULBP-2/5/6-PerCP (165903, R&D Systems), CD54-PB (HCD54, Biolegend), B7H6-APC (875001, R&D Systems), ecto-calreticulin-A488 (EPR3924, Abcam), MICA/B-PE/Cy7 (6D4, Biolegend), CD20-PE (2H7, BioLegend), CD38-A488 (HIT2, BioLegend), FAS-FITC (DX2, Biolegend), DR4-PE (DJR1, Biolegend), DR5-APC (DJR2-4, Biolegend), HLA-E-PE/Cy7 (3D12, Biolegend) and total HLA class I proteins (W6/32, Biolegend) for 30 min at 4 °C. Assessment of CLL cells that have recently egressed from the lymph nodes was performed as previously described [30 (link), 31 (link)]. Patient CLL cells were rested for 1 h at 37 °C, then stained with CD19-BV510 (HIB19), CD5-APC, HLA-E-PE/Cy7 (3D12), pan-HLA (W6/32) and CXCR4-PE (12G5, Biolegend). Cells were then acquired on a BD FACS Aria II.
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