Primary hTM cells were cultured on plastic dishes or glass coverslips to confluency (approximately 90%) in 10% fetal bovine serum growth media. Cells were subsequently serum starved for 24 hours, after which respective treatment with vehicle control (veh), LPA (20 µM; catalog number: 10010093; Cayman Chemical, Ann Arbor, MI, USA), IL6 (100 ng/mL; catalog number: SRP3096; Sigma Aldrich, St. Louis, MO, USA)/sIL6R (200 ng/mL; catalog number: SRP3097; Sigma Aldrich, St. Louis, MO, USA), or both (LPA + IL6/sIL6R) in serum-free media was done for 24 hours. In another set of experiments, the aforementioned treatments were performed in the presence or absence of 2 µM verteporfin (YAP inhibitor, without light stimulation using aluminum foil; catalog number: 17334; Cayman Chemical, Ann Arbor, MI, USA) or 2 µM STAT3 inhibitor (Catalog number: 573097; Sigma Aldrich, St. Louis, MO, USA) in serum-free media for 24 hours. The concentration of verteporfin used in this study has previously been verified to be safe and efficacious in hTM cells and other ocular/nonocular cells.57 (link)–59 (link) Herein, we determined a safe and effective dose for the STAT3 inhibitor (that is, 2 µM) by performing a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Supplementary Fig. S1 ) and Western blotting.
Sil6r
Manufactured by Merck Group
Sourced in United States
The SIL6R is a high-performance laboratory instrument designed for precise and reliable analytical measurements. It features advanced sensing technologies and robust construction to ensure accurate and reproducible results. The core function of the SIL6R is to provide reliable data collection and analysis capabilities for scientific research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Merck Group (2 mentions)
Sgp130, by Merck Group (2 mentions)
Sicam1, by Merck Group (2 mentions)
Svcam1, by Merck Group (2 mentions)
Stnfrii, by Merck Group (2 mentions)
Stat3 inhibitor, by Merck Group (1 mentions)
Verteporfin, by Cayman Chemical (1 mentions)
Igfbp 1, by Merck Group (1 mentions)
Igfbp 3, by Merck Group (1 mentions)
Igfbp2, by Merck Group (1 mentions)
C reactive protein (crp), by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Arachidonic acid, by Merck Group (1 mentions)
Puromycin dihydrochloride, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (1 mentions)
240 b 002 cf, by R&D Systems (1 mentions)
Hep3b, by Merck Group (1 mentions)
4 protocols using sil6r
1
Modulation of hTM Cell Signaling
2
Multiplex Measurement of Serum Immune and Growth Factors
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Fluorescent bead‐based immune‐assays for IL1Ra, IL8, MCP‐1, MIP‐1β, CRP, SAA, MMP1, MMP2, MMP9, sgp130, sICAM1, sVCAM1, sIL2Rα, sIL6R, sTNFRI, sTNFRII, sEGFR, IGFBP1, IGFBP2, IGFBP3, insulin‐like growth factors binding protein‐6 (IGFBP6) and tPAI1 (Millipore Inc.), were used to measure serum levels. Briefly, serum samples were incubated with antibody‐coated microspheres, followed by biotinylated detection antibody. Detection of the proteins was accomplished by incubation with phycoerythrin‐labeled streptavidin. The resultant bead immuno‐complexes were then read on a FLEXMAP3D (Luminex) with the instrument settings recommended by the manufacturer.
The captured median fluorescence intensity (MFI) data was subjected to our quality control steps. Briefly, wells with individual bead counts below 30 or bead count coefficient of variation (CV) above 200 were flagged for exclusion. Replicate wells with CV ≥ 25% were excluded from further analyses. The standard concentration and MFI were log2 transformed before regression. Protein concentrations were estimated using a regression fit to the standard curve with serial dilution of known concentration for each protein.
12 (link),
13
The captured median fluorescence intensity (MFI) data was subjected to our quality control steps. Briefly, wells with individual bead counts below 30 or bead count coefficient of variation (CV) above 200 were flagged for exclusion. Replicate wells with CV ≥ 25% were excluded from further analyses. The standard concentration and MFI were log2 transformed before regression. Protein concentrations were estimated using a regression fit to the standard curve with serial dilution of known concentration for each protein.
12 (link),
13
3
Multiplex Luminex Immunoassay for Inflammatory Markers
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Luminex immunoassays for sTNFR-I, sTNFR-II, CRP, SAA, sIL6R, sgp130, sICAM-1, and sVCAM1 were obtained from Millipore (Millipore Inc., Billerica, MA, USA). Multiplex immunoassays were performed according to the manufacturer's instructions. Briefly, serum samples were incubated with antibody-coated microspheres, followed by biotinylated detection antibody. Proteins were detected by incubation with phycoerythrin-labeled streptavidin and the resultant bead immunocomplexes were read on a FLEXMAP3D (Luminex, TX, USA) with the following instrument settings: events/bead: 50, minimum events: 0, flow rate: 60 μL/min, sample size: 50 μL, and discriminator gate: 8000–13500. Median fluorescence intensity (MFI) was collected and used for calculating protein concentration.
4
Stimulation Protocols for Cytokine and Fatty Acid Signaling
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For stimulation experiments, the cells were seeded on 6-well plates and grown until 80% confluency was reached. The cells were then starved in DMEM containing 0.5% FBS for 16 h. Stimulations were completed in DMEM containing 0.2% FBS. For the treatments, 10 ng/mL of recombinant human TNF-α (#210-TA-005/CF, R&D Systems, Wiesbaden, Germany), 2.5 ng/mL of recombinant human TGF-β (#240-B-002/CF, R&D Systems), 2.5 ng/mL of recombinant human IL-1β (#HZ-1164, Miltenyi Biotech., Bergisch Gladbach, Germany) and indicated concentrations of IL-6 (Miltenyi Biotech.) were used for the indicated times. In order to analyze IL-6 trans-signaling, 100 ng/mL of soluble IL6-Rα human (sIL-6R, #SRP3097, Sigma-Aldrich) was used. To inhibit potential signaling pathways downstream of IL-6, cells were pretreated for 1 h with either JAK inhibitor Ruxolitinib (5 µM, #HY-50856), STAT3 inhibitor STATTIC (10 µM, # HY-13818) or PI3K inhibitior LY294002 (10 µM, #HY-10108), all obtained from MedChem Express (Hölzel Diagnostika Handels GmbH, Cologne, Germany). Puromycin-dihydrochloride was obtained from Sigma-Aldrich (#P8833). The fatty acids oleic acid (#O3008), palmitic acid (#P0500), arachidonic acid (#10931) and linoleic acid (#L1376) used for IL-6 co-stimulation experiments in Hep3B were all obtained from Sigma Aldrich and used at indicated concentrations.
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