For flow cytometry analysis, 2x105 isolated cells per sample were incubated in 100 µl MACS buffer for 20 minutes in the dark with different sets of antibodies, as specified in Supplementary Table 1
Lsrfortessa x 20 device
Manufactured by BD
Sourced in Germany
The BD LSRFortessa™ X-20 is a flow cytometer designed for advanced multiparameter analysis. It features a compact design and is capable of detecting up to 20 parameters simultaneously.
Automatically generated - may contain errors
2 protocols using lsrfortessa x 20 device
1
Flow Cytometry Protocol for Immunophenotyping
2
Isolation and Characterization of Myeloid-Derived Suppressor Cells
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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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