Staining for sorting was performed using cryo-preserved PBMCs resuspended in PBS supplemented with 2% FBS and 2 mM EDTA. Cells were stained for 30 min at 4°C with CD71-FITC (clone CY1G4), CD19-PE (clone HIB19), CD38-BV605 (clone HIT2), CD20-APC-Fire750 (clone 2H7), and Zombie Aqua (all from BioLegend). Cells were then washed twice, and single plasmablasts (live singlet CD19+ CD38+ CD71+) were sorted with a MoFlo (Beckman-Coulter) into 96-well plates containing 10 μL of 10 mM Tris supplemented with 1 U/μL RNase inhibitor (Promega), and immediately frozen on dry ice.
Cd19 pe clone hib19
Manufactured by BioLegend
CD19-PE (clone HIB19) is a fluorochrome-conjugated monoclonal antibody that specifically binds to the CD19 antigen expressed on the surface of B cells. It is suitable for the identification and enumeration of B cells in flow cytometry applications.
Automatically generated - may contain errors
2 protocols using cd19 pe clone hib19
1
Sorting and Freezing Plasmablasts
2
Multiparametric Flow Cytometry Analysis
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The efficiency of gene transfer to total CD34+ cells and the CD45RA− subset thereof was determined after staining the cells with the following anti-human-specific antibodies (all from eBioscience, unless noted otherwise): CD34-APC (clone 8G12, STEMCELL Technologies), and CD45RA-APC780 (clone HI100).
For phenotypic analysis of human cells from NSG mice, flow-cytometric analysis was performed on freshly collected BM cells. Cells were treated with red blood cell lysis buffer (STEMCELL Technologies), washed, and incubated with a blocking reagent (PBS with 2% fetal bovine serum (FBS), 5% human serum, and anti-CD16/CD32 antibody [2.4G2]). The cells were then stained with the following anti-human-specific antibodies: CD45-Alexa Fluor 700 (clone HI30, BioLegend), CD33-PECY7 (clone WM-53, eBioscience), CD19-PE (clone HIB19, BioLegend), CD20-PE (clone L27, StemCell Technologies). A minimum of 200,000 BM cells were analyzed per mouse. A negative control was set on non-transplanted mouse BM. In some cases, we utilized a fluorescence-minus-one (FMO) gating strategy to overcome the ambiguity created by artifacts that could be introduced by simultaneously compensating different fluorochromes.
For phenotypic analysis of human cells from NSG mice, flow-cytometric analysis was performed on freshly collected BM cells. Cells were treated with red blood cell lysis buffer (STEMCELL Technologies), washed, and incubated with a blocking reagent (PBS with 2% fetal bovine serum (FBS), 5% human serum, and anti-CD16/CD32 antibody [2.4G2]). The cells were then stained with the following anti-human-specific antibodies: CD45-Alexa Fluor 700 (clone HI30, BioLegend), CD33-PECY7 (clone WM-53, eBioscience), CD19-PE (clone HIB19, BioLegend), CD20-PE (clone L27, StemCell Technologies). A minimum of 200,000 BM cells were analyzed per mouse. A negative control was set on non-transplanted mouse BM. In some cases, we utilized a fluorescence-minus-one (FMO) gating strategy to overcome the ambiguity created by artifacts that could be introduced by simultaneously compensating different fluorochromes.
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