PBMCs were isolated using Ficoll-Paque and stained with Live/dead Fixable Aqua dead cell stain kit 405 nm (life technologies), CD3-PB (UCHT1, BD), CD14-PB (M5E2, BD), CD19-APC-Cy7 (SJ25C1, BD), CD20-AF700 (2H7, Biolegend) and CD27-PE-Cy7(M-T271, BD). Antigen-specific staining was performed using CCP2-BV605, CCP2-APC and CArgP2-PE, or HC55-APC and HC55-PE [25 (link)] or TT-APC and TT-PE. Antigen-specific single B cell sorts were performed on a BD FACSAria III 4 L cell sorter at the Flow cytometry Core Facility (FCF) of the Leiden University Medical Center (LUMC) in Leiden, Netherlands. The cells were cultured on irradiated CD40L L cells in complex RMPI medium for 10–12 days [26 (link)]. RNA isolation, cDNA production, ARTISAN PCR and sequencing was performed as previously described [27 (link), 28 (link)].
Facsaria 3 4 l cell sorter
Manufactured by BD
The BD FACSAria III 4 L cell sorter is a high-performance cell sorting instrument. It features a 4-laser, 12-color configuration designed for advanced multiparameter analysis and sorting of complex samples.
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2 protocols using facsaria 3 4 l cell sorter
1
Isolation and Culture of Antigen-Specific B Cells
2
Stable GFP-MDC1 Cell Line Generation
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For generation of the GFP-MDC1 expressing stable cell lines, U2OS ΔMDC1 cells were transfected with the indicated plasmids using Lipofectamine 2000, following the standard protocol provided by the supplier. The media was changed 10 h post-transfection. Two days post-transfection, cell colonies were grown in the presence of 400 μg/mL Geneticin G418 for 10 days with a media change every 3 days. Single colonies were picked using cloning rings and expanded. Clones were analyzed for GFP-MDC1 expression by western blotting and immunofluorescence, respectively. To improve homogeneous GFP-MDC1 expression, positive clones were sorted using a BD FACSAria III 4L cell sorter.
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