CIA was induced by immunization of type II collagen (C II) and complete Freund’s adjuvant (CFA) in DBA/1J mice as previously described5 (link) and described in greater detail below. On day 14 after initial CII immunization, 2 × 106 GMSCs were intravenously injected into the CIA. B7-H1high GMSCs and B7-H1low GMSC subsets were sorted by flow cytometry sorter and 107 cells/mL were suspended in in PBS and then injected into the CIA mice. GMSC were pretreated with B7-H1 blocking antibody (Biolegend, 10 μg/mL), rh-IFN-γ (Biolegend, 10 ng/mL), or STAT-3 inhibitor (SELLECK, 10 μM) and washed twice with RPMI 1640 (Hyclone) containing 10% heat-inactivated fetal bovine serum (Hyclone), 100 IU/mL penicillin (GIBCO), 1% sodium pyruvate (Corning), and 1% HEPES (Corning) before the GMSC were used in the in vitro and in vivo experiments described below. GMSC that had been used at each time point were obtained from different donors and mice in each experimental time interval received the same cell population from the same donor. Five mice were used in each group and the animal experiments were repeated at least three times.
Rhifn γ
Manufactured by BioLegend
RhIFN-γ is a recombinant non-glycosylated form of rhesus macaque interferon gamma. Interferon gamma is a cytokine that plays a crucial role in immune system regulation and host defense against infections.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
B7 h1 blocking antibody, by BioLegend (1 mentions)
Stat3 inhibitor, by Selleck Chemicals (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
Hepes, by Corning (1 mentions)
Fixable live dead stain, by Thermo Fisher Scientific (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
Rhil 12, by BioLegend (1 mentions)
Biotinylated goat f ab 2 anti human λ κ, by Southern Biotech (1 mentions)
3 protocols using rhifn γ
1
GMSC Therapy Alleviates Collagen-Induced Arthritis
2
Activation of B Cell Signaling Pathways
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Tonsil B cells (1 × 106) were cultured in complete RPMI for 40 h in 8-well chambers containing PLB without any stimulation or with PLB-Ag, rhIFN-γ (50 ng/ml, Biolegend) and CpG (ODN2006) (1 μM, Invivogen). In vitro cultured cells were harvested, washed with RPMI 1640 supplemented with 0.1% heat-inactivated FBS and rested for 60 min at 37°C. For surface staining, cells were incubated with anti-IgD Fab fragment (Southern Biotech), anti-CD10 antibody (HI10a) from BD Biosciences and live/dead fixable stain (Invitrogen) for 20 min at RT and washed with RPMI 1640 supplemented with 0.1% heat-inactivated FBS. F(ab′)2 anti-human IgG + IgA + IgM (H+L) (Jackson ImmunoResearch) was added to the cells at a final concentration of 10 μg/ml and incubated at 37°C for 5 min, followed by fixation with 4% PFA for 10 min at 37°C. Phospho-proteins of Syk, BLNK, Igα, and PLCγ2 were analyzed by flow cytometry.
3
Tonsil B Cell Activation by BCR and TLR9
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Tonsil B cells (1 × 106) were stimulated with various combinations of BCR and TLR9 ligand, in presence of cytokines, IFN-γ and IL-12+IL-18. For BCR stimulation, 10 nM biotinylated goat F(ab′)2 anti-human λ + κ (Southern biotech) was used for either soluble Ag or membrane-bound Ag on PLB or PMS. B cells were cultured in complete RPMI at 37°C for 40 h in 8-well chambers containing either PLB-Ag or PMS-Ag for membrane Ag engagement and B cells were cultured in round-bottomed 96-well plates for soluble antigen engagement. When needed, cells were supplemented with CpG (ODN2006) (1 μM, Invivogen) in culture media for TLR9 stimulation. For cytokines, rhIFN-γ (50 ng/ml, Biolegend), rhIL-12 (50 ng/ml, Biolegend), and rhIL-18 (50 ng/ml, MBL) were used.
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