Cd19 alexa fluor 700

The following anti-human antibodies were used for staining: CD68-FITC, CD14-PercP-Cy5.5, HLA-DR-APC, CD15-PE-Cy7, CD11b-PErcP-Cy5.5, CD163-BV421, CD14-APC, HLA-DR-PE, CD33-BV421 and PD-L1-BV421 purchased from Biolegend (San Diego, CA), CD11b-PE, CD3-Alexa Fluor 700, CD19-Alexa Fluor 700, CD19-Alexa Fluor 700, CD163-Alexa Fluor 647, CD16-PE-Cy7, CD3-PE, CD19-PE and CD56-PE purchased from BD Biosciences (San Jose, CA), CD14-PE-TR and CD16 PE-TR purchased from Life Technologies (Carlsbad, CA) and CD86-FITC purchased from R&D systems (Minneapolis, MN). CD32-a-FITC Ab was purchased from Stemcell technologies, (Vancouver, Canada). CD-32-B (F-4) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, and secondary Ab anti-mouse F(ab’)2 was purchased from Thermo fisher scientific (MA, USA).
Intracellular staining of CD68 was performed as follows: PBMC were stained with surface marker antibodies, fixed with fixation/permeabilization buffer (eBioscience), washed, and stained for intracellular antigens in 1X permeabilization buffer. Cells were analyzed on an LSR Fortessa (BD) flow cytometer, and data analyzed using Flow Jo (Treestar, Ashland, OR). Dead cells were excluded based on viability dye staining (Zombie Aqua Fixable Viability Dye, Biolegend).

Flow cytometry was used to follow changes in classical B cell immunophenotype markers (CD19, IgD, IgM, and CD27) as well as those involved in energy pathways including CD24, CD39, CD73, and CD38 throughout in vitro culture. B cell numbers, B cell growth (total mass), and viability were also calculated using flow cytometry. Briefly, PBMCs or B cells were stained for 20 min with fluorescent conjugates of monoclonal antibodies to CD19-Alexa Fluor 700, IgD-BV421, and IgM-BV605 (BD Biosciences, San Jose, CA, USA), CD27-APC and CD24-APC eFluor780 (eBioscience, San Diego, CA, USA), CD39-FITC, CD73-PE, and CD38-PerCP.Cy5.5 (Biolegend, San Diego, CA, USA), and a viability marker (LIVE/DEAD™ Fixable aqua dead cell stain, ThermoFisher Scientific, Waltham, MA, USA). The cells were washed and resuspended in PBS and acquired within 24 h on a BD LSR FortessaTMX-20. Total mass was calculated by combining the mass of debris, dead cells, and live cells in each culture well using the following formula: (FSC × mean number viable + FSC × mean number dead + FSC × mean number debris). Compensation beads (BD, Biosciences, San Jose, CA, USA) were used to optimize the fluorescence compensation settings for multicolor flow cytometric analysis. A minimum of 100,000 events in the lymphocyte gate was collected.