PBMC samples were thawed in a 37°C water bath, and cells were immediately and gently added to pre-chilled thaw diluent (1:30 ratio) containing Plasma-Lyte A Injection, pH 7.4 (Baxter Healthcare Corp) with added heparin (10 IU/mL, StemCell Tech) and DNase I (0.01 mg/mL, Stem Cell Tech). Cells were centrifuged at 300x g for 5 min at room temperature and pellet resuspended in 1X RoboSep buffer (StemCell Tech) for subsequent B cell isolation. If cell aggregates were observed, cell suspensions were filtered with a 37 μm cell strainer (StemCell Tech). B cell isolation was performed using the EasySep Human B cell Isolation kit (StemCell Tech) with minor adjustments to the standard protocol, including performing isolation in a 96-well plate with proportionately scaled-down reagents. B cell purity was assessed via flow cytometry for select samples. Cells were stained with: CD19 PE (BD; clone HIB19), CD14 FITC (BD; clone M5E2), CD15 BV510 (BD; clone W6D3), CD3 BV421 (BD; clone SK7), CD56 PE-Cy7 (BD; clone B159), CD45 APC-Cy7 (BD; clone 2D1), and 7-AAD (BioLegend). Samples were acquired on a CytoFlex analyzer (Beckman Coulter), and data were analyzed using FlowJo v10 (FlowJo, LLC).
Cd15 bv510
Manufactured by BD
The CD15 BV510 is a fluorescent-labeled antibody used for the identification and enumeration of CD15-positive cells in flow cytometry analysis. It binds to the CD15 antigen, which is expressed on the surface of granulocytes, monocytes, and some lymphocytes. The BV510 fluorescent dye is used to label the antibody, allowing for its detection and quantification by flow cytometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
37 μm cell strainer, by STEMCELL (1 mentions)
Dnase 1, by STEMCELL (1 mentions)
Cytoflex analyzer, by Beckman Coulter (1 mentions)
Easysep human b cell isolation kit, by STEMCELL (1 mentions)
7 aad, by BioLegend (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
Robosep buffer, by STEMCELL (1 mentions)
Cd3 bv421, by BD (1 mentions)
Cd19 pe, by BD (1 mentions)
Heparin, by STEMCELL (1 mentions)
Cd45 apc cy7, by BD (1 mentions)
Lsrfortessa x 20 device, by BD (1 mentions)
Sepmate 50 tube, by STEMCELL (1 mentions)
Cd14 bv421, by BD (1 mentions)
Hla dr percp, by BD (1 mentions)
Facsaria 3 instrument, by BD (1 mentions)
2 protocols using cd15 bv510
1
Isolating B Cells from Cryopreserved PBMCs
2
Isolation and Characterization of Myeloid-Derived Suppressor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Heparin-containing tubes were used to collect peripheral blood samples from healthy donors and cancer patients. Then, PBMC were separated from the peripheral blood by density gradient centrifugation method using Ficoll-Paque Plus medium and SepMate-50 tubes (STEMCELL Technologies).
The PBMC were stained for 20 min at 4°C with the following anti-human antibodies: lineage marker (Lin) (CD3, CD19, CD20, CD56 PE [BD Biosciences]), HLA-DR PerCP (BD Biosciences), CD11b BV710 (BD Biosciences), CD33 BB515 (BD Biosciences), CD14 BV421 (BD Biosciences), and CD15 BV510 (BD Biosciences). Isotype-matched antibodies were used as controls. Flow cytometric analysis was performed using a BD LSRFortessa X-20 device, and cell sorting was performed using a FACSAria III instrument (BD Biosciences). The data were analyzed with FlowJo software (BD Biosciences). We used the established phenotypes for the MDSC analysis.[ 111617181920212223 ] CD11b + CD33 + Lin -HLA-DR -/low cells were defined as total MDSC. Then, the total MDSC were divided into three subsets including M-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 + CD15 -), G-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 -CD15 + ), and I-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 -CD15 -).
The PBMC were stained for 20 min at 4°C with the following anti-human antibodies: lineage marker (Lin) (CD3, CD19, CD20, CD56 PE [BD Biosciences]), HLA-DR PerCP (BD Biosciences), CD11b BV710 (BD Biosciences), CD33 BB515 (BD Biosciences), CD14 BV421 (BD Biosciences), and CD15 BV510 (BD Biosciences). Isotype-matched antibodies were used as controls. Flow cytometric analysis was performed using a BD LSRFortessa X-20 device, and cell sorting was performed using a FACSAria III instrument (BD Biosciences). The data were analyzed with FlowJo software (BD Biosciences). We used the established phenotypes for the MDSC analysis.[ 111617181920212223 ] CD11b + CD33 + Lin -HLA-DR -/low cells were defined as total MDSC. Then, the total MDSC were divided into three subsets including M-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 + CD15 -), G-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 -CD15 + ), and I-MDSC (CD11b + CD33 + Lin -HLA-DR -/low CD14 -CD15 -).
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