Anti-mouse CD3e and CD28 antibodies [monoclonal antibodies (mAbs)] were purchased from eBiosciences. Recombinant mouse IL-4, TNF-α, and human TGF-β were purchased from R&D Systems. Anti-TNFR1 (Monoclonal Hamster IgG Clone # 55R170) and anti-TNFR2 (Monoclonal Hamster IgG Clone # TR7589.29) blocking antibodies and control immunoglobulin G were purchased from R&D Systems. STAT5 inhibitor was purchased from Santa Cruz. Nuclear factor-κB (NF-κB) inhibitors bortezomib, CAS 213546-53-3, and JSH-23 were purchased from Selleckchem. CFSE (carboxyl-fluorescein diacetate, succinimidyl ester) was purchased from Invitrogen. The OVA (257–264, SII NFE KL) peptide used in the OT-I mouse model was purchased from GL Biochem (Shanghai) Ltd.
Stat5 inhibitor
Manufactured by Santa Cruz Biotechnology
Sourced in United States
STAT5 inhibitor is a laboratory reagent designed to inhibit the activity of the STAT5 protein. STAT5 is a transcription factor involved in cellular signaling pathways. This inhibitor can be used in research applications to study the role of STAT5 in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Recombinant mouse il 4, by R&D Systems (2 mentions)
Human tgf β, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Control immunoglobulin g, by R&D Systems (1 mentions)
Jsh 23, by Selleck Chemicals (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Anti ifn γ, by BD (1 mentions)
Macs separation column, by Miltenyi Biotec (1 mentions)
Anti cd3, by BD (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Fty720, by Merck Group (1 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
Lps escherichia coli o127 b8, by Merck Group (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Recombinant human il 7, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Anti cd14 magnetic bead, by Miltenyi Biotec (1 mentions)
7 protocols using stat5 inhibitor
1
Immune Cell Stimulation Reagents
2
Th17 Cell Differentiation from Splenic CD4+ T Cells
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To purify splenic CD4+ T cells, splenocytes were incubated with anti-CD4-coated magnetic beads. CD4+ T cells were isolated using magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec). Cells were cultured in the presence of plate-bound anti-CD3 and soluble anti-CD28 (each at a concentration of 1 µg/mL; both from BD PharMingen), anti-interferon γ (anti-IFN-γ), and anti-IL-4 (each at a concentration of 5 µg/mL) for 3 days. Th17 cell differentiation was induced by treatment with IL-6 (20 ng/mL) and transforming growth factor β (TGF-β; 2 ng/mL). The cells were treated by STAT5 inhibitor (Santa Cruz) to reduce STAT5 expression.
3
Modulating Immune Cell Signaling Pathways
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FTY720 (Sigma) was used to block S1PR1. WT or Rag1−/− mice were injected intraperitoneally (i.p.) with 20 μg FTY720 every 24 h for 6 days. Rag1−/− mice were injected i.p. with 100 μg STAT5 inhibitor (Santa Cruz Biotechnology) every 24 h for 3 days to inhibit STAT5 signaling. To block IL-7Rα signaling, Rag1−/− mice were injected i.p. with 200 μg IL-7Rα blockade antibody (A7R34, Bio X Cell), using single or continuous injections, as indicated.
4
Isolation and Culture of Human Monocytes
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PBMCs of anonymous healthy donors were isolated from buffy coats purchased from the Beijing Red Cross Blood Center using density gradient cell separation by Ficoll (Lymphoprep; STEMCELL Technologies) following the protocol approved by the institutional review board of the School of Medicine, Tsinghua University. The private information of anonymous blood donors was inaccessible to investigators. PBMCs of RA patients were obtained from Peking Union Medical College Hospital using the protocol that was approved by the institutional review board of Peking Union Medical College Hospital. CD14+ monocytes were further isolated from PBMCs using anti-CD14 magnetic beads (130-050-201; Miltenyi Biotec). CD14+ monocytes were cultured in RPMI 1640 medium (10040CM; Corning) supplemented with 10% (vol/vol) FBS (Gibco) and human recombinant M-CSF (300-25, 10 ng/ml; PeproTech). LPS (Escherichia coli O127:B8; Sigma-Aldrich), human recombinant IL-7 (200-07; PeproTech), human recombinant TNF (H8916; Sigma-Aldrich), or chemical inhibitors (SB203580 from Selleck; STAT5 inhibitor from Santa Cruz Biotechnology; and Bay 11-7082 from Sigma-Aldrich) were used as indicated for various experiments.
5
Isolation and Treatment of Pancreatic Islets
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Intact pancreatic islets were isolated from mice as previously described.8 (link) Briefly, mouse pancreas was given intra-ductal injection of 0.3 mg/ml collagenase P (Roche, Mannheim, Germany) in Hanks' balanced salt solution (Sigma-Aldrich). The pancreas was removed and incubated in 37 °C for 15 min. After washing and gradient centrifugation, the islets were handpicked under a stereomicroscope. Isolated islets were then cultured overnight in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% (vol/vol) bovine fetal serum (Gibco, Grand Island, NY, USA), 1% (vol/vol) penicillin, and streptomycin (Life Technologies). Islets were treated with recombinant FGF21 (University of Hong Kong; HKU, Hong Kong, China),50 (link) recombinant GH (R&D Systems, Minneapolis, MN, USA), rosiglitazone (Sigma-Aldrich) or STAT5 inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
6
Culturing and Stimulating Human Bronchial Epithelial Cells
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The human bronchial epithelial cell line, 16HBEo- (16HBE; Shanghai Fuxiang Biological Technology Co. Ltd., ATCC, USA) was grown in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (Gibco) and placed in a humidified incubator at 37 °C with an atmosphere of 5% CO2. When the cells reached 80–90% confluence, the cells were treated with trypsin and seeded into culture plates at a density of 104–105 cells per cm2 for use in the experiments. The medium was changed to serum-free RPMI 1640 when the cells reached 85% confluence, and after 12 h, the cells were rinsed with PBS and then stimulated with HDM for the indicated times and doses. The cells were also treated with other mediators and inhibitors, namely lfTSLP, the extracellular signal-regulated kinase (ERK)1/2 tyrosine kinase inhibitor U0126 (10 μM; Cell Signaling Technology, USA), the p38 kinase inhibitor SB203580 (10 μM; Cell Signaling Technology), the STAT5 Inhibitor (10 μM; Santa Cruz, USA), sfTSLP, and 1,25D3 for 1 h prior to stimulation with HDM and lfTSLP in serum-free medium.
7
Modulating Immune Cell Responses
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Recombinant mouse IL-4, TNF-α and human TGF-β were purchased from R&D Systems. CFSE (carboxylfluorescein diacetate, succinimidyl ester) was purchased from Invitrogen. Functional anti-mouse CD3e and CD28 antibodies (mAbs) were purchased from eBioscience. Anti-TNFR1 and anti-TNFR2 blocking mAbs and control IgG were purchased from Biolegend. STAT5 inhibitor and Bortezomib (a NF-κB inhibitor) were purchased from Santa Cruz and Selleckchem respectively.
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