The adoptive B cell transfer experiments were performed at the age of 8 weeks (both donor and recipient mice). At this age, the immune system of the mice is fully developed [28 (link)]. B-1 cells were isolated from the peritoneal cavity of the wild-type mice by negative selection with magnetic-activated cell sorting (MACS) cell separation [29 (link)]. First, B cells were isolated with the Pan B Cell Isolation Kit II (130-104-443, Miltenyi Biotec, Bergisch Gladbach, Germany). Next, B-2 cells were excluded using CD23 MicroMeads (130-098-784, Miltenyi Biotec) and peritoneal macrophages excluded with anti-F4/80 MicroBeads UltraPure (130-110-443, Miltenyi Biotec) to obtain the B-1 cells. The B-2 cells were isolated from the spleen of wild-type mice with negative selection strategy by excluding B-1 cells using CD43 MicroBeads (130-049-801, Miltenyi Biotec) from the B cells isolated with the Pan B Cell Isolation Kit II [30 (link)]. B cell-deficient JHT mice were transferred with 5 × 106 B-1 or B-2 cells, respectively [30 (link)]. Two weeks after B cell transfer, the aortae were isolated from the recipient mice for the myograph experiments.
Pan b cell isolation kit 2
Manufactured by Miltenyi Biotec
Sourced in Germany
The Pan B Cell Isolation Kit II is a lab equipment product designed for the isolation of B cells from a cell suspension. It utilizes magnetic bead-based separation technology to effectively enrich for B cells.
Automatically generated - may contain errors
Lab products found in correlation
Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Cd8a isolation kit 2, by Miltenyi Biotec (2 mentions)
Cd4 isolation kit 2, by Miltenyi Biotec (2 mentions)
Macrophage isolation kit, by Miltenyi Biotec (1 mentions)
Pan dc isolation kit, by Miltenyi Biotec (1 mentions)
Ack buffer, by Beyotime (1 mentions)
Percoll gradient, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
X vivo medium, by Lonza (1 mentions)
Multimacs cell24 separator plus, by Miltenyi Biotec (1 mentions)
Pharm lyse, by BD (1 mentions)
Anti cd19 pe antibodies, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Complete dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
B6 sjl ptprca pepcb boyj mice, by Jackson ImmunoResearch (1 mentions)
Anti cd3 145 2c11, by Thermo Fisher Scientific (1 mentions)
Anti cd28 37.51, by Thermo Fisher Scientific (1 mentions)
Naive cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
17 protocols using pan b cell isolation kit 2
1
Adoptive Transfer of B Cell Subsets
2
Isolation of Immune Cell Subsets
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T cells, B cells, NK cells, and DCs were isolated from the spleen using a Pan T Cell Isolation Kit II, a Pan B Cell Isolation Kit II, a Macrophage Isolation Kit, and a Pan DC Isolation Kit, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). Neutrophils were isolated from bone marrow cells using the Percoll gradient (Sigma-Aldrich, St. Louis, MO, USA), as described elsewhere (37 (link), 38 (link)). Briefly, bone marrow cells were extracted from mouse femurs and tibias. After removal of red blood cells by the ACK buffer (Beyotime, Shanghai, China), cells were carefully loaded on a 52, 69, 78% percoll (GE Healthcare) gradient, and centrifuged at 1500 g for 30 min without breaking. Cells were isolated on the 69/78% interface layer. In some experiments, peritoneal neutrophils were isolated after CLP surgery. Pasteur pipettes were filled with cold PBS and inserted through the peritoneal wall to inject PBS into each mouse. After aspiration of the fluid from the peritoneum, the peritoneal fluid was slowly withdrawn. The cell suspension was then centrifuged at 1700 rpm for 5 min and washed with PBS.
3
Murine Splenic B Cell Stimulation
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Murine splenic B cells were purified using the Pan B Cell Isolation Kit II (130-104-443, Miltenyi). Cells were cultured at a density of 1 million/ml supplemented with 10 μg/ml LPS (Santa Cruz) for stimulation. Medium was replaced on day 3 of culture by replacing half volume with fresh culture medium containing LPS followed by analysis on day 4 and day 5. For 4-OHT-inducible Tfeb knockout B cells, cells were stimulated with 10 ng/mL murine recombinant IL-4 (214-14, PeproTech) and 5 μg/mL anti-mouse CD40 antibody (Thermo Fisher) for 4 days.
4
Acetate Modulates Arthritis Progression
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Arthritis was induced on day 0 using 4 mg (200 μl) of ArthritoMab Monoclonal Antibody Cocktail for C57BL/6 administered i.p. (CIA-MAB-2C, MD Biosciences). Two hours later, 200 μl of 106 acetate-stimulated B cells or 106 nonstimulated B cells resuspended in PBS plus 2% FBS or 200 μl of PBS plus 2% FBS were injected i.p. On day 3, 50 μg of LPS (0.5 mg/mL) were administered i.p. Joints were blindly monitored daily by 2 investigators and mice were culled on day 7. To assess the effect of acetate, acetate was administered in drinking water at 200 mM, 3 weeks prior and throughout the experiment. For B10 cell adoptive transfer experiments, on day 2, peritoneal cells were cultured overnight in the presence or absence of acetate (10 mM). On day 1, B cells from nonstimulated and acetate-stimulated peritoneal cells were sorted using Pan B Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions.
5
Isolation of Splenic B Cells from Irradiated Mice
6
Isolation and Coculture of Antibody-Secreting B Cells
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To isolate antibody-secreting B cells, splenic single-cell suspensions from rhFVIII-injected HemA mice were generated as described above. Subsequently, cells were centrifuged for 5 minutes at 1,500 rpm at 4°C and cleared from red blood cells by hemolysis using a hypotonic red cell lysis buffer (146 mM NH4Cl, 10 mM NaHCO3, and 2 mM EDTA). Hemolysis was stopped with RPMI 1640 (Life Technologies) media supplemented with 2% FCS. After centrifugation, cells were handled according to the manufacturer’s instructions (Miltenyi Biotec, Pan B cell isolation Kit II). Finally, 0.3 × 105 preactivated B cells were cocultured with 0.5 × 105 WT Tregs (isolated as described above). To block PD-1 signaling, an inhibitory PD-1 antibody (RPM1-14; 40 μg/mL) was added to the coculture. Coculture was performed in X-Vivo medium (Lonza) supplemented with 10% FCS, 100 U/mL penicillin, 100 U/mL streptomycin, and 5.5 × 10–5 M β-mercaptoethanol in a 96-well plate (TPP) and restimulated with 2.5 μg rhFVIII at 37°C and 5% CO2 overnight.
7
Enrichment of Murine Splenic B Cells
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Spleens were harvested at the indicated time points of the immunization schedule. Spleen cell suspensions were recovered following disassociating using a 40 µm cell strainer. Cellular suspensions were pelleted at 300 g for 5 min, and red blood cell lysis was performed for 1 min using BD Pharm Lyse (BD). Cells were washed twice with MACS buffer and re-suspended in 3 ml of MACS buffer. These cells were further processed according to the manufacturer’s protocol using the Pan B Cell Isolation Kit II (Miltenyi) on a MultiMACS Cell24 Separator Plus (Miltenyi, program depletion). Purity of B-cell lineage was usually above 90% (data not shown).
8
Isolation and Characterization of B Cells
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Peripheral blood samples from study subjects were collected in heparinized tubes and immediately processed. Peripheral blood mononuclear cells (PBMCs) were isolated over Lymphocyte Separation Medium (LSM 1077) gradients for immediate use. Cells were kept at 4°C or on ice and cold buffers were employed to minimize alterations in gene expression during labeling and isolation. The purified B cells were prepared from PBMCs using negative selection by Pan B Cell Isolation Kit II from Miltenyi Biotec (Bergisch Gladbach, Germany). Immunofluorescence labeling for flow cytometry was performed by incubating isolated B cell with anti-CD19-PE antibodies (BD Biosciences, San Jose, CA) for 30 minutes and washing. Flow cytometry was performed using a FACSCalibur (BD Biosciences) with CellQuest software. Analysis was conducted with FloJo software. The purification of B cells over 95% was used in the study.
9
Isolation and Culture of Immune Cell Subsets
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Mouse splenic CD4+ T cells, CD8+ T cells, B cells were isolated using a CD4 isolation kit II (Miltenyi Biotec), CD8a isolation kit II (Miltenyi Biotec) and PanBcell isolation kit II (Miltenyi Biotec) by negative selection, respectively. A purity rate of >96.6% for isolated CD4+ T cells, CD8+ T cells and B cells were confirmed by flow cytometry. Peritoneal macrophages were obtained from B6 and MRL mice by washing their peritoneal cavities with 15 ml of ice-cold saline. The cells from individual mice were centrifuged at 1500 rpm for 5 minutes at 4 °C, washed in complete DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin, and subsequently adjusted to 1 × 107 cells/ml. The cells were cultured on 12-well flat-bottom tissue culture plates (Corning) and incubated for 3 hours at 37 °C under 5% CO2 air.
10
Adoptive Transfer of B Cells
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For experiments male B6.SJL-Ptprcapepcb/BoyJ mice (CD45.1+/+) 8–12 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME). CD45.2+ B cells from male or female 09–1B12-UCA KI mice (H09–1B12-UCA/WT, κ09−1B12-UCA/WT) were enriched using the Pan B Cell Isolation Kit II (Miltenyi Biotec), counted, diluted to desired cell numbers in PBS and adoptively transferred into CD45.1+ recipient mice as reported previously43 (link). Ages of the recipient animals ranged from 8–10 weeks.
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