NSC676914A (Additional file 1 : Figure S1A) was synthesized by diazotization of 2-[(4-aminophenyl)sulfonyl]-1-hydrogen sulfate followed by coupling with m-toluidine. An unsulfated alcohol analog (Additional file 1 : Figure S1B) was similarly prepared from 2-[(4-aminophenyl)sulfonyl]-1-ethanol. Both compounds were purified to >95% purity by reverse phase HPLC. DMSO stocks were used for all experiments. IKKβ inhibitor (IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide, catalog #401484) (Additional file 1 : Figure S1C) was purchased from EMD Biosciences (La Jolla, CA). LPA (857130C) was purchased from Avanti Polar Lipids (Alabaster, AL). LPA stocks were made in PBS containing 1% fatty acid-free bovine serum albumin. TPA (4174) was purchased from Cell Signaling Technology, Inc (Danvers, MA). Recombinant Human TNFα (300-01A) was purchased from Peprotech (Rocky Hill, NJ). Z-VAD-FMK (2163) and Necrostatin-1 (2324) were purchased from Tocris Bioscience (Ellisville, MO). Puromycin, XTT, PMS, LPS (L5293) and N-Acetyl-L-cysteine (A7250) were purchased from Sigma-Aldrich (St. Louis, MO).
Lps l5293
Manufactured by Merck Group
Sourced in United States
LPS (L5293) is a laboratory product manufactured by Merck Group. It serves as a lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. The core function of this product is to facilitate research and analysis related to bacterial cell structure and immune system responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd40 antibody, by BioLegend (2 mentions)
Macs magnetic beads, by Miltenyi Biotec (2 mentions)
Z vad fmk 2163, by Bio-Techne (1 mentions)
N acetyl l cysteine a7250, by Merck Group (1 mentions)
Recombinant human tnf α 300 01a, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
A2383, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Corticosterone, by MedChemExpress (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by MedChemExpress (1 mentions)
5 protocols using lps l5293
1
Synthesis and Characterization of IKKβ Inhibitors
2
B Cell Stimulation Protocol
3
B Cell Stimulation Protocol
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B cells were isolated by CD19 positive selection, using MACS magnetic beads (130-052-201, Miltenyi Biotec, Bergisch Gladbach, Germany). The B cell purity was confirmed by double staining of CD19 and CD45R using fluorescence-activated cell sorting. The 105 cells per well were stimulated with 5 μg/ml lipopolysaccharides (LPS; L5293, Sigma-Aldrich) and/or 10 μg/ml anti-CD40 antibody (102810, BioLegend, San Diego, CA, USA). Supernatants were harvested 48 hours later.
4
Propagation and Differentiation of THP-1 Cells
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The maintenance and propagation of HEK293T cells have been previously described [43 (link)]. Human monocytic THP-1 cells, purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea), were cultured in RPMI 1640 (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum, 1% penicillin/streptomycin and 0.05mM 2-mercaptoethanol. THP-1 cells were differentiated by incubation with 100 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA) for 15 h, followed by replacement with fresh medium without PMA. The cells were incubated with 500 ng/mL LPS (L5293, Sigma-Aldrich) and 5 mM ATP (A2383, Sigma-Aldrich) to active NLRP3 inflammasomes. Cell supernatants were concentrated 30-fold using Amicon Ultra Centrifugal Filters 0.5 mL 10K (Millipore, Burlington, MA, USA). Transient transfection using Omicsfect™ was performed according to the manufacturer’s instructions (Omics Bio, Taipei city, Taiwan).
5
Cell Stress Model and Pharmacological Intervention
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The PC-12 cell line was purchased from the National Collection of Authenticated Cell Cultures (China, Shanghai) and the HAPI cell line from BeNa Culture Collection (Beijing, China). PC-12 cells were cultured in RPMI 1640 medium (Gibco, USA) and the HAPI cells in the DMEM/F-12 medium (Gibco). All culture systems were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco) under a humidified atmosphere of 5% CO2 at 37 °C.
The cell treatment of the experiments is depicted in Figs.6 and 7 and was as follows: for the LPS/corticosterone + Rg1 group, the cells were pre-treated with LPS (1 μg/mL, to establish an inflammatory stress model) or corticosterone (400 μM, to establish an oxidative stress model) for 2 h, followed by co-incubation with Rg1 (5 μM, 10 μM, 20 μM) for 24 h. For the LPS/corticosterone + shRNA group, the cells were transfected with lentivirus for 48 h and then washed with cold PBS buffer, followed by incubation with LPS or corticosterone for 24 h. corticosterone (HY-B1618) was purchased from MedChemExpress and LPS (L5293) from Sigma-Aldrich.
The cell treatment of the experiments is depicted in Figs.
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