Heparinized blood samples were obtained from normal donors and patients enrolled in clinical research protocols approved by the Human Subjects Protection Committee of the Dana-Farber Cancer Institute (DFCI), at UCSD and the Mayo Clinic (CLL Research Consortium), and through the ICGC (42 (link)) after obtaining written informed consent. Treatment indication for all 21 patients in the discovery cohort was determined based on iwCLL criteria (12 (link)), (44 (link)). Peripheral blood mononuclear cells (PBMC) from normal donors and patients were isolated by Ficoll/Hypaque density gradient centrifugation. Mononuclear cells were used fresh or cryopreserved with 10% DMSO FBS and stored in vapor-phase liquid nitrogen until the time of analysis. CD19+ B cells from normal volunteers and CLL samples with WBC ≤50 × 109/L were isolated by immunomagnetic selection (Miltenyi Biotec, Auburn CA) and stained with anti CD19 PE (BioLegend) prior to FACS sorting for live single cells in the presence of DAPI. MBL cells and naïve and memory B cells from age-matched healthy donors were isolated as follows: cryopreserved PBMCs were thawed and stained with anti CD19 PE, CD5 FITC, and CD27 APC (BioLegend). Cells were gated for naïve B cells (CD19+; CD27-; CD5), memory B cells (CD19+; CD27+; CD5-) or MBL (CD19+; CD5+) (Supplementary Figure 7a ).
Anti cd19 pe
Manufactured by BioLegend
Sourced in United States
Anti-CD19-PE is a monoclonal antibody conjugated with the fluorescent dye phycoerythrin (PE). It is designed for the detection and analysis of CD19-expressing cells, which are primarily B lymphocytes, using flow cytometry.
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Lab products found in correlation
Anti cd4 fitc, by BioLegend (5 mentions)
Anti cd38 fitc, by BioLegend (3 mentions)
Anti cd45 efluor450, by Thermo Fisher Scientific (2 mentions)
Anti cd3 apc, by Thermo Fisher Scientific (2 mentions)
Anti cd8 pe cy7, by BioLegend (2 mentions)
7 aad, by BioLegend (2 mentions)
Anti cd138 brillantviolet605, by BioLegend (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti cd27 pe, by BioLegend (2 mentions)
Anti cd43 fitc, by BioLegend (2 mentions)
Anti cd34 pe, by BioLegend (2 mentions)
Anti cd90 fitc, by BioLegend (2 mentions)
Anti cd45 bv421, by BioLegend (2 mentions)
Anti cd3 pe cy7, by BioLegend (2 mentions)
Anti cd4 percp cy5, by BioLegend (2 mentions)
Anti cd3 percp cy5, by BioLegend (2 mentions)
Anti cd45 apc, by BioLegend (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
22 protocols using anti cd19 pe
1
Isolation and Characterization of B Cells
2
Flow cytometric analysis of bladder immune cells
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Bladders were aseptically removed, minced with scissors, and digested at 37° for 30 minutes in RPMI-1640 with 10mM HEPES, collagenase D (C5318, Sigma-Aldrich), and DNAse (10104159001, Sigma-Aldrich). Bladders were forced through a 70 μm cell strainer (352350, Corning) and washed with 5% FBS in PBS. Single cell suspensions were stained with anti-CD45-eFluor450 (48–0451-82, eBioscience), anti-CD3-APC (17–0032-82, eBioscience), anti-CD19-PE (115511, BioLegend), anti-CD4-FITC (100405, BioLegend), anti-CD8-PE/Cy7 (100721, BioLegend), anti-CD138-BrillantViolet605 (142515, BioLegend), and 7-AAD (420404, BioLegend). Data was acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0. Gates were determined with isotype antibodies in bladder suspensions from young mice.
3
Characterization of B-cell Differentiation
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LN biopsy samples were minced and evaluated by two-color flow cytometry after staining in phosphate-buffered-saline without calcium chloride magnesium chloride (PBS (−)) with the following monoclonal antibodies: anti-CD19-phycoerythrin (PE) (BioLegend, San Diego, CA, USA), anti-CD27-PE (BioLegend), anti-CD3-PE (BioLegend), anti-CD20-fluorescein isothiocyanate (FITC) (BioLegend), and anti-CD38-FITC (BioLegend). BiPSCs were also evaluated by two-color flow cytometry before and after the induction of hematopoietic differentiation using the following monoclonal antibodies: anti-CD19-PE, anti-CD27-PE, anti-CD34-PE (BioLegend), anti-CD20-FITC, anti-CD38-FITC, anti-CD43-FITC (BioLegend), and anti-CD45-FITC (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a BD FACSCANTO II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
4
Multiparametric Flow Cytometry of Infiltrating Immune Cells
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In in vivo experiments, mice were sacrificed after tMCAO. The BALF was centrifuged at 500 g to collect cells. After being washed with PBS, cells were fixed and permeabilized (Invitrogen, Intracellular Fixation & Permeabilization Buffer Set), then stained with intracellular antibodies. The following antibodies were used: anti‐CD45‐BV421 (Biolegend, 103 134, clone: 30‐F11, 1:400), anti‐ F4/80‐AF488 (Biolegend, 123 120, clone: BM8, 1:400), anti‐LY6G‐APCCy7 (Biolegend, 108 424, clone: RB6‐8C5, 1:400), anti‐CD11c‐APC (Biolegend, 117 310, clone: N418, 1:400), anti‐CD3‐PECy7 (Biolegend, 100 220, clone: 17A2, 1:400), anti‐CD19‐PE (Biolegend, 152 408, clone: 1D3/CD19, 1:400), anti‐CD45‐PerCPCy5.5 (Biolegend, 368 504, clone: 2D1, 1:400), anti‐CD90‐FITC (Biolegend, 328 108, clone: 5E10, 1:400), anti‐CD34‐APC (Biolegend, 343 509, clone: 581, 1:400), and anti‐CD29‐PE (Biolegend, 303 004, clone: TS2/16, 1:400). Cell viability of BMDM was assessed using the Annexin V‐APC/PI apoptosis Detection Kit (KeyGEN, KGA1030‐100) according to manufacturer' s instructions.
5
Phenotypic Characterization of CD34+ Cells
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Purified CD34+ cells were evaluated by two-color and three-color flow cytometry after staining in phosphate-buffered saline without calcium chloride or magnesium chloride with the following monoclonal antibodies: anti-CD19-PE, anti-CD34-PE (BioLegend, San Diego, CA, USA), anti-CD38-FITC, anti-CD43-FITC, anti-CD10-FITC (BioLegend), and anti-CD45-PE/Cy5 (BioLegend). Immunofluorescence of the labeled cell membrane was evaluated using a flow cytometer (S3e Cell Sorter; BIO-RAD). Furthermore, phenotype analysis was performed after co-culture of CD34+ cells with MS-541 (link).
6
Immunophenotyping of Mouse and Human Cells
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For mouse cell phenotype analysis, anti CD3-percp/cy5.5, anti CD4-FITC, anti CXCR5-allophycocyanin, anti ICOS-PE, and anti PD-1-PE were purchased from BioLegend (San Diego, CA, USA). Anti CD19-FITC, anti CD138-PE, anti IgD-allophycocyanin, anti CD27-percp/cy5.5, and relevant IgG isotypes were purchased from eBioscience (San Diego, CA, USA).
For human cell analysis, anti CD3-FITC, anti CD4-percp/cy5.5, anti CXCR5-allophycocyanin, anti PD-1-PE, and anti CD19-PE were purchased from BioLegend. Anti-IL-21 neutralizing antibody (eBioscience) and anti-CD40 (BioLegend) were used for functional analysis in vitro.
For human cell analysis, anti CD3-FITC, anti CD4-percp/cy5.5, anti CXCR5-allophycocyanin, anti PD-1-PE, and anti CD19-PE were purchased from BioLegend. Anti-IL-21 neutralizing antibody (eBioscience) and anti-CD40 (BioLegend) were used for functional analysis in vitro.
7
Multicolor Flow Cytometry Analysis of Immune Cells
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Cells were washed in flow cytometry staining buffer (BD Pharmingen), treated with Fc block (BD Bioscience) and stained for flow cytometric analyses of extracellular markers using fluorochrome‐conjugated antibodies according to the manufacturer's instructions. Antibodies included anti‐CD19‐PE or anti‐CD19‐FITC, anti‐CD5‐BV421 and the activation markers anti‐HLA‐DP, DQ, DR Alexa Fluor 647 or anti‐HLA‐DP/DQ/DR‐FITC, anti‐CD86‐PerCP‐CY5.5, anti‐CD69‐FITC and anti‐CD40‐PE‐Cy7 (BioLegend); markers of maturation anti‐CD38‐FITC or anti‐CD38‐APC and CD24‐APC‐Cy7 (BioLegend); or CD22‐FITC and sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐10‐APC (BioLegend). Immunoglobulin isotype‐matched control antibodies were used to confirm specificity of staining. Extracellular staining was conducted for 30 min in the dark at 4°C, before the cells were washed and analysed by flow cytometry. To correct for inter‐assay variation, ratios were calculated of staining intensity in treated versus control cultures.
8
Characterizing MSC Surface Markers and Apoptosis
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To characterize the expression of surface markers, the MSCs were immune-stained and detected by flow cytometry analysis. Antibodies used in this detection are anti-CD73-PE, anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD19-PE, anti-CD45-APC and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
9
Characterization of Leukemia and Lymphoma Cell Lines
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Unless otherwise specified, all cell lines and reagents were procured from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Human acute lymphoblastic leukemia cell lines (BALL-1, REH, SUP; B15, NALM-6, CCRF, and Jurkat) as well as the human Burkitt lymphoma cell lines Raji and Daudi were cultured in RPMI-1640 medium (Gibco Invitrogen, USA). The culture medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological Industries, IL) and 1% Penicillin–Streptomycin Solution (Gibco Invitrogen). Cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2.
One million tumor cells were harvested from the culture, washed twice in cold Phosphate Buffer Saline (PBS; Gibco Invitrogen), and then pelleted at 1000 rpm for 5 min. Surface expression of CD19 and CD20 antigens on different tumor cells was detected by staining with human block Fc-receptors (2.5 μL, BioLegend, San Diego, USA) for 20 min at 4 °C, followed by a single wash and subsequent staining with anti-CD19-PE (5 μL, BioLegend, clone: H1819) or anti-CD20-APC antibodies (5 μL, BioLegend, clone:2H7) for 30 min at 4 °C. Non-staining tumor cells served as negative controls. After two washes, cells were resuspended in 200 μL PBS for quantitative analysis by flow cytometry.
One million tumor cells were harvested from the culture, washed twice in cold Phosphate Buffer Saline (PBS; Gibco Invitrogen), and then pelleted at 1000 rpm for 5 min. Surface expression of CD19 and CD20 antigens on different tumor cells was detected by staining with human block Fc-receptors (2.5 μL, BioLegend, San Diego, USA) for 20 min at 4 °C, followed by a single wash and subsequent staining with anti-CD19-PE (5 μL, BioLegend, clone: H1819) or anti-CD20-APC antibodies (5 μL, BioLegend, clone:2H7) for 30 min at 4 °C. Non-staining tumor cells served as negative controls. After two washes, cells were resuspended in 200 μL PBS for quantitative analysis by flow cytometry.
10
Comprehensive CAR Expression Analysis
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For evaluation of CAR expression, cells were stained with a goat anti-mouse Fab antibody conjugated with Alexa Fluor 647 (Jackson ImmunoResearch) or biotinylated CD19-Fc (ACRO Biosystems) for 15 min at room temperature. Cells were thoroughly washed before staining with antibodies for additional surface markers. Anti-CD3-BB515 and anti-CD62L-BB515 were purchased from BD Biosciences. Anti-CD4-BV785, anti-CD8-BV711, anti-CD45RA-BV421, anti-CD45RO-PECy7, streptavidin-PE, anti-CD27-PE, anti-CD95-allophycocyanin (APC)Cy7, anti-CCR7-APC, anti-CD45-BV711, and anti-CD19-PE, anti-CD3-BV711 were purchased from BioLegend and used to stain surface antigens. Cell Trace Violet was purchased from Thermo Fisher Scientific and used at a concentration of 1 μM to label cells for 10 min. All data were acquired using a BD Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software.
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