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.
B7 h1 blocking antibody
Manufactured by BioLegend
The B7-H1 blocking antibody is a laboratory tool designed to interfere with the interaction between the B7-H1 protein and its receptor. B7-H1 is a key immunoregulatory molecule involved in modulating immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Rhifn γ, by BioLegend (1 mentions)
Stat3 inhibitor, by Selleck Chemicals (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
Hepes, by Corning (1 mentions)
Cisplatin, by BioLegend (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Anti cd28, by BioLegend (1 mentions)
2 protocols using b7 h1 blocking antibody
1
GMSC Therapy Alleviates Collagen-Induced Arthritis
2
T-Cell Proliferation Assay with Cisplatin-Treated A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole blood was collected from healthy individuals at the Suzhou Blood Center
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.
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