Surface staining was done by incubating fresh PBMC with a cocktail of antibodies containing 5 μl CD19 PerCP (Clone 4G7, BD, Catalog No. 345778), 3 μl CD21 PE-CY7 (Clone B-ly4, BD, Catalog No. 561374), 20 μl CD27 FITC (Clone Ll28, BD, Catalog No. 340424), 3 μl CD23 BV421 (Clone M-L233, BD, Catalog No. 562707), 10 μl sIgD PE(Clone IA6–2, BD, Catalog No. 555779), 10 μl sIgM APC (Clone G20–127, BD, Catalog No. 551062) in panel one and 5 μl CD19 PerCP (Clone 4G7, BD, Catalog No. 345778), 3 μl CD21 PE-CY7 (Clone B-ly4, BD, Catalog No. 561374), 20 μl CD27 FITC (Clone Ll28, BD, Catalog No. 340424), 10 μl CD 5 PE(Clone Ll7F12, BD, Catalog No. 345782), 3 μl CD38 APC (Clone HB7, BD, Catalog No. 345807) and 3 μl CD24 BV421 (Clone ML5, BD, Catalog No. 562789) in panel two. Incubations were done for 20 min at room temperature in the dark. Then cells were washed and re-suspended in FACS (Fluorescence Activated Cell Sorting) buffer. Data were acquired on a BD FACSCanto II with Diva software and analyzed using FlowJo software (Version 9.6.2).
Cd19 percp
Manufactured by BD
Sourced in United States
The CD19-PerCP is a fluorescently labeled antibody that binds to the CD19 surface antigen, which is expressed on B cells. It is a tool used in flow cytometry applications for the identification and enumeration of B cells in biological samples.
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Lab products found in correlation
Cd3 fitc, by BD (5 mentions)
Cd45 amcyan, by BD (3 mentions)
Cd34 pe cy7, by BD (3 mentions)
Cd10 pe, by BD (3 mentions)
Cd14 percp, by BD (3 mentions)
Cd4 fitc, by BD (3 mentions)
Cellquest software, by BD (3 mentions)
Imatinib, by Selleck Chemicals (2 mentions)
Ruxolitinib, by Selleck Chemicals (2 mentions)
Cd8 fitc, by BD (2 mentions)
Cd21 apc, by BD (2 mentions)
Ifn γ pe, by Thermo Fisher Scientific (2 mentions)
Via probe, by BD (2 mentions)
Facssort machine, by BD (2 mentions)
Cd4 pe, by BD (2 mentions)
Cd8 apc, by BD (2 mentions)
Cd38 fitc, by BD (2 mentions)
Pd 1 pe, by BD (2 mentions)
Cd8 percp, by BD (2 mentions)
Cd38 apc, by BD (2 mentions)
18 protocols using cd19 percp
1
Comprehensive B-cell Immunophenotyping
2
Pathway Analysis of Pediatric ALL
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To identify Jak2/Stat5, ABL, and Ras pathway alterations in ALL patient samples, fresh cells from bone marrow were stimulated with 200 ng/ml thymic stromal lymphopoietin (TSLP) (PeproTech, Cranbury, NJ, USA) for 30 min at 37 °C, and stained with the following cell surface monoclonal antibodies: CD45 Amcyan, CD34 PECy7, CD19 PerCP, and CD10 PE (Becton Dickinson, USA) [32 ]. Then, the cells were fixed with BD Phosflow Fix Buffer I, permeabilized with BD Phosflow Perm Buffer III (Becton Dickinson), and incubated with monoclonal antibodies to identify phosphorylated targets of each pathway: Stat5 (pY694) Pacific Blue to identify Jak2, CrkL (pY207) AF488 for ABL, and Erk 1/2 (pT202/pY204) AF488 for Ras. In cases with an adequate number of cells, the abnormal pathway activity was selectively inhibited in vitro for 30 min with ruxolitinib (5 nm ) (Selleckchem, Houston, TX, USA) to inhibit Jak2/Stat5 pathway, and with imatinib and/or dasatinib (5 μm ) (Selleckchem) to prevent ABL abnormal activation. The antibody concentration and conditions for each assay were performed based on the manufacturer's recommendations. Flow cytometry was performed using a BD FACSVerse Cell Analyzer System and data were analyzed by FlowJo vX software.
3
Detecting Oncogenic Pathway Activation
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To detect Jak2-Stat5 pathway activation, fresh cells from bone marrow were stimulated with 200 ng/mL of thymic stromal lymphopoietin (TSLP) (PeproTech, Cranbury, NJ, USA) for 30 min at 37 °C, and stained with the following cell surface monoclonal antibodies: CD45 Amcyan, CD34 PECy7, CD19 PerCP, and CD10 PE (Becton Dickinson, Franklin Lakes, NJ, USA). Then, the cells were fixed and incubated with monoclonal antibodies, as previously reported, to identify the phosphorylated target Stat5 (pY694) Pacific Blue. For Abl and Ras pathways, similar conditions were used, except for the TSLP stimulation; the surrogate marker CrkL (pY207) AF488 was used for Abl, and Erk 1/2 (pT202/pY204) AF488 for Ras; a positive result was considered when populations >5% of blasts were detected. In samples with adequate numbers of cells, abnormal pathway activity was selectively inhibited in vitro for 30 min with ruxolitinib (5 nM) (Selleckchem, Houston, TX, USA) to inhibit the Jak2-Stat5 pathway, and with imatinib (5 μM) (Selleckchem) to prevent Abl abnormal activation. The antibody concentration and conditions for each assay were performed based on the manufacturer’s recommendations. Flow cytometry was performed using a BD FACSVerse Cell Analyzer System and data were analyzed by FlowJo vX software.
4
Isolation and Identification of T-cell Subsets
5
Flow Cytometric Analysis of CRLF2 Expression
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In patients from whom cells were available, flow cytometry studies were performed. CRLF2 protein expression was assessed using an allophycocyanin (APC)‐conjugated monoclonal antibody that recognizes the extracellular domain (Becton Dickinson, Franklin Lakes, NJ, USA) [32 ]. Bone marrow cells were stained with cell surface monoclonal antibodies: CD45 Amcyan, CD34 PECy7, CD19 PerCP, and CD10 PE (Becton Dickinson). To identify cell surface CRLF2 or intracellular CRLF2, two different staining conditions were used: CRLF2 antibody incubation in fresh cells and CRLF2 antibody incubation after fixation, and permeabilization with BD Cytofix/Cytopermä (Becton Dickinson). The antibody concentration and conditions for the assay were performed as per the manufacturer's recommendations. Flow cytometry was performed using a BD FACSVerse Cell Analyzer System and data were analyzed by FlowJo vX software (Becton, Dickinson and Company, Ashland, OR, USA).
6
Immunophenotyping of Murine Macrophages
7
Comprehensive Immunophenotyping of T and B Cells
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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).
8
Enrichment Protocol for T-cell Frequency Analysis
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Direct analysis of T-cell frequency was accomplished using our previously published enrichment approach (12 (link)). Briefly, 30–50 × 106 PBMCs were resuspended in 200 µL of T-cell media, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 μg/mL phycoerythrin (PE)-labeled tetramer at room temperature for 100 min, followed by antibody staining with CD4-APC (eBioscience), CD45RO-FITC (eBioscience), and a combination of CD14-PerCP and CD19-PerCP (BD Biosciences) for 15 min at 4°C. Cells were washed, incubated with PE magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and enriched with a magnetic column, retaining 1% of the cells as a nonenriched sample. The PE-enriched and precolumn samples were labeled with Via-Probe (BD Biosciences) and analyzed on a FACSCalibur (BD Biosciences), gating on CD4+CD14−CD19−Via-Probe− and plotting tetramer versus CD45RO. Frequencies were calculated as previously described (12 (link)). Statistical analysis was performed using unpaired t tests with Welch correction with Prism software (version 5.03, GraphPad Software Inc.).
9
Immunophenotyping of T-cell subsets in HSCT recipients
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Peripheral blood samples were collected from recipients on days 30, 60, and 90 after HSCT. The samples were stained without further separation to minimize selective loss shortly after collection. The combinations of the directly conjugated monoclonal antibodies CD3-FITC, CD4-PE, CD8-APC, CD19-Per-CP, CD25-PE (BD Biosciences, Mountain View, CA, USA), and their isotype-matched antibodies were used to analyze the immunophenotype of T lymphocyte subsets. Flow cytometry was performed using a BD FACSSort machine (Becton Dickinson Biosciences, San Jose, CA, USA). The data were analyzed using CellQuest software (BD Biosciences).
10
Annexin V Apoptosis Detection in Immune Cells
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Apoptosis was detected using Annexin V apoptosis detection kit FITC (e-Bioscience; catalog number 88-8005-72) per manufacturer’s instructions. Briefly, after harvesting THP-1 and PBMC at the indicated times, cells were centrifuged and washed once with cold PBS followed by another wash with 1X cold Annexin V Binding Buffer. To the cells (106 cells/100μl), 5μL of fluorochrome-conjugated Annexin V was added and incubated in the dark for 15 min. Cells were washed once with 1X Annexin V Binding Buffer and re-suspended in 300μL of 1X Annexin V Binding Buffer containing 5μL Propidium iodide (eBioscience). Percent apoptosis in PBMC subsets (T cells, B cells, dendritic cells, and monocytes) was determined by staining with antibodies (BD Bioscience) specific for immune cell surface markers: HLA-DR-allophycocyanin-Cy7 (Catalog number 335796), CDllc-APC (Catalog number 340544), CD19-PerCP (Catalog number 340421), CD14-Alexa Fluor 700 (Catalog number 557923), CD3-PE-Cy7 (Catalog number 563423). Samples were analyzed using an LSRII flow cytometer (BD Bioscience).
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