Cells were incubated in 50 μL of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD). Antibodies included anti-IgM FITC (G20-127, BD), anti-CD19 BUV395 (SJ25C1, BD), anti-CD3 BV711 (UCHT1, BD), anti-CD14 BV711 (M0P-9, BD), anti-CD16 BV711 (3G8, BD), anti-CD20 BUV737 (2H7, BD), anti-IgD BV605 (IA6-2, BD), and a fixable viability dye (Tonbo Biosciences, cat#13-0870-T500). B cells were individually sorted into flatbottom 96-well plates containing feeder cells that had been seeded at a density of 28,600 cells/well one day prior in 100 µL of IMDM media (Gibco, cat#31980030) containing 10% fetal calf serum, 100 U/mL penicillin plus 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin. These 3T3 feeder cells produce CD40L, IL2, and IL21 to stimulate antibody secretion into culture supernatants [19 (link)]. B cells sorted onto feeder cells were cultured at 37 °C for 13 days.
Anti cd14 bv711 m0p 9
Manufactured by BD
Anti-CD14 BV711 (M0P-9) is a laboratory reagent that can be used for the detection and analysis of CD14, a protein expressed on the surface of certain immune cells. It is a fluorescently labeled antibody that binds to CD14 and can be used in flow cytometry and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd16 bv711 3g8, by BD (4 mentions)
Anti igm fitc g20 127, by BD (3 mentions)
Facsaria, by BD (2 mentions)
Imdm media, by Thermo Fisher Scientific (2 mentions)
Anti cd19 buv395 sj25c1, by BD (2 mentions)
Anti cd3 bv711 ucht1, by BD (2 mentions)
Anti cd20 buv737 2h7, by BD (2 mentions)
Anti igd bv605 ia6 2, by BD (2 mentions)
Newborn calf serum, by Thermo Fisher Scientific (2 mentions)
Anti cd19 bv421 hib19, by BD (2 mentions)
Anti cd45 bv510 hi30, by BD (2 mentions)
Anti cd3 bv605 ucht1, by BioLegend (2 mentions)
Facsymphony, by BD (1 mentions)
Anti cd8 pe rpa t8, by BD (1 mentions)
Fixable viability stain 700, by BD (1 mentions)
Facsymphony a5, by BD (1 mentions)
4 protocols using anti cd14 bv711 m0p 9
1
B Cell Stimulation and Antibody Production
2
Quantifying B Cells via Flow Cytometry
3
Quantification of B Cells and Plasma Cells
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B cells were quantified using a research flow cytometry panel (Representative gates shown in Extended Data Fig. 8 ). Briefly, peripheral blood mononuclear cells (PBMCs) were incubated on ice for 30 minutes with fluorescently labeled antibodies (in fluorescence-activated single cell sorting [FACS] buffer containing Dulbecco's phosphate-buffered saline [DPBS] and 1% newborn calf serum [Life Technologies]) before its analysis on a FACSymphony A5 (BD Bioscience) flow cytometer. The following antibodies were included for cell labeling: fixable viability stain 700 (BD cat#564997), anti-CD19 BV421 (HIB19, BD), anti-CD45 BV510 (HI30, BD), anti-CD3 BV605 (UCHT1, BioLegend), anti-CD16 BV711 (3G8, BD), anti-CD14 BV711 (M0P-9, BD). FlowJoTM Software version 10.7.1 was used for analyses with flow cytometry proportions multiplied by absolute lymphocyte counts to calculate total CD19+ B cell numbers. Plasma cells in the bone marrow were quantified based on detection of CD138+ plasma cells in clinically obtained bone marrow core biopsies by immunohistochemistry in the University of Washington Hematopathology Laboratory.
4
Isolation and Culture of B Cells
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Cells were incubated in 50 μL of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD). Antibodies included anti-IgM FITC (G20–127, BD, cat#555782, 1:80 dilution), anti-CD19 BUV395 (SJ25C1, BD, cat#563551, 1:20 dilution), anti-CD3 BV711 (UCHT1, BD, cat#563725, 1:50 dilution), anti-CD14 BV711 (M0P-9, BD, cat#563372, 1:50 dilution), anti-CD16 BV711 (3G8, BD, cat#563127, 1:50 dilution), anti-CD20 BUV737 (2H7, BD, cat#612849, 1:20 dilution), anti-IgD BV605 (IA6–2, BD, cat#563313, 1:50 dilution), anti-CD27 PE/Cy7 (LG.7F9, eBioscience, cat#25-0271-82, 1:160 dilution), and a fixable viability dye (Tonbo Biosciences, cat#13-0870-T500, 1:250 dilution). B cells were individually sorted into either 1) empty 96-well PCR plates and immediately frozen, or 2) flat bottom 96-well plates containing feeder cells that had been seeded at a density of 28,600 cells/well one day prior in 100 µL of IMDM media (Gibco, cat#31980030) containing 10% fetal calf serum, 100 U/mL penicillin plus 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin. B cells sorted onto feeder cells were cultured at 37 °C for 13 days.
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