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2 protocols using pe cy7 conjugated cd19

1

Multi-Parameter Immune Cell Profiling

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For human B cell staining, cells were stained with PE-Cy7-conjugated anti-CD19 (BioLegend, #302216), PE-conjugated CD27 (BioLegend, #302808), BV421-conjugated IgD (BioLegend, #348226), BV421-conjugated anti-CD138 (BioLegend, #356516), and APC-conjugated anti-IgG antibodies (BioLegend, #304136). Alternately, cells were permeabilized and stained with primary antibodies against ORAI1, ORAI2, and STIM2 at 37°C for 30 min. Cells were then washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor® 488 Goat antirabbit IgG (Thermo Fisher, #A-11008) secondary antibodies at room temperature for 30 min. For mouse cell staining, cells were stained with APC-conjugated B220 (BioLegend, #318326), PE-Cy7-conjugated CD38 (BioLegend, #102718), BV421-conjugated CD138 (BioLegend, #142523), PE-conjugated PD-1 (BioLegend, #135205), APC-conjugated CXCR5 (BioLegend, #145506), PE-conjugated CD95 (BioLegend, #152608), fluorescein isothiocyanate (FITC)-conjugated GL-7 (BioLegend, #144612), APC-Cy7-conjugated-CD4 (BioLegend, #100526), BV510-conjugated-CD3 (BioLegend, #100234), Percp-cy5.5-conjugated-CD45 (BioLegend, #103132), APC-conjugated-CD8 (BioLegend, #100712), PE-Cy7-conjugated-CD19 (BioLegend, #506921) and BV605-conjugated-CD11b (BioLegend, #101257). Samples were analyzed using a BD LSR Fortessa (BD Bioscience).
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2

Multiparameter Flow Cytometry of Murine and Humanized Immune Cells

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Tumors, spleens, and lymph nodes of treated mice were collected, smashed through a 70 μm cell strainer and cultured over night in 10% RPMI-1640 medium. Single cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies and analyzed using a FACSAria flow cytometer (BD Biosciences) or Gallios flow cytometer (Beckman Coulter, Brea, CA) and FlowJo 7.6 software (TreeStar, Ashland, OR). Anti-mouse FITC-conjugated CD8 (Proimmune), PE-Cy7-conjugated CD19 (BioLegend), APC-conjugated CD4 (BioLegend), PE-Cy7-conjugated CD3e (BioLegend), PE-conjugated IFNgamma (BioLegend) and APC-conjugated SIINFEKL MHC-I pentamer (Proimmune, Oxford, UK) were used for immunocompetent mouse experiment. Anti-human FITC-conjugated CD45RA (BioLegend), PE-conjugated CD197 (BioLegend), PerCP-conjugated CD8 (BioLegend), PE-TexasRed-conjugated CD4 (LifeTechnologies), FITC-conjugated IFNgamma (BioLegend), PE-conjugated IL-6 (BioLegend), and APC-conjugated TNFα (BioLegend) were used for humanized mouse experiment.
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