Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Application (200P-05N) and cultured in EGM-2MV complete medium (Lonza, CA94045-820) or M200 complete medium (Thermo Fisher, M200500; S00310) until they reached ~95–100% confluence. To generate conditioned medium, cells were exposed to serum-free medium (apoptotic stress; RPMI 1640, Invitrogen, 11875-093) alone or in the presence of hydrogen peroxide (necrotic stress; 15 mM, Fischer, H325-500), bafilomycin A1 (lysosomal inhibitor; 20 nM, Sigma-Aldrich, B1793, Baf), wortmannin (PI3K3 inhibitor; 100 nM, Sigma-Aldrich, W3144, Wort), ZVAD-fmk (pancaspase inhibitor; 50 μM, R&D Systems), DEVD (caspase-3/-7 inhibitor; 100 μM, R&D Systems), or vehicle (dimethyl sulfoxide, Sigma-Aldrich, D2650) for 1 to 4 h. Preincubation for 1 h in complete medium with wortmannin was applied before serum starvation. Cells were exposed to EGM-2MV vesicle-free complete medium to produce conditioned medium from normal cells.
B1793
Manufactured by Merck Group
Sourced in United States
B1793 is a laboratory equipment product manufactured by Merck Group. It serves as a basic multi-purpose tool for various scientific and research applications. The core function of B1793 is to provide a reliable and versatile platform for conducting experiments and analyses in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
C6628, by Merck Group (4 mentions)
M9281, by Merck Group (3 mentions)
C2759, by Merck Group (3 mentions)
A7250, by Merck Group (2 mentions)
Y0001976, by Merck Group (2 mentions)
D3695, by Merck Group (2 mentions)
Aob13386, by Merck Group (2 mentions)
Sml0743, by Cell Signaling Technology (2 mentions)
Cas 338967 87 6, by Merck Group (2 mentions)
M8699, by Merck Group (2 mentions)
M200500, by Thermo Fisher Scientific (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
D2650, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
E2888, by Merck Group (1 mentions)
D6546, by Merck Group (1 mentions)
M8046, by Merck Group (1 mentions)
Bafa1, by Merck Group (1 mentions)
14 protocols using b1793
1
HUVEC Conditioned Media Production
2
Cell Culture and Starvation Protocol
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Cells were cultured in DMEM (D6546; Sigma-Aldrich) supplemented with 10% FBS (172012; Sigma-Aldrich), 50 U/ml penicillin, 50 µg/ml streptomycin (15070-063; Gibco), and 2 mM glutamine (25030-081; Gibco; regular medium) in a 5% CO2 incubator. For starvation treatment, cells were washed twice with PBS and cultured in Earle’s balanced salt solution (E2888; Sigma-Aldrich) or amino acid–free DMEM (048-33575; Wako) without FBS. For bafilomycin A1 treatment (B1793; Sigma-Aldrich), cells were cultured with 100 nM bafilomycin A1 for 2 h.
3
Cell Culture and Inhibitor Treatments
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Mouse hepatoma Hepa1-6 cells, human cervical carcinoma HeLa cells, human hepatoma HepG2 cells, and human lung carcinoma A549 cells were purchased from the ATCC. The cells were cultured in DMEM (25 mM glucose, 11995040, Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% HEPES (BOSTER, PYG0019) at 37 °C and 5% CO2. GFP-LC3/HeLa cells were maintained in DMEM (25 mM glucose), and supplemented with 0.4 mg/mL G418 (10131035, Thermo Scientific, Waltham, MA, USA) at 37 °C and 5% CO2.
For inhibitory assays, the antagonists or inhibitors, such as RU486 (30 μM, M8046, Sigma-Aldrich, St. Louis, MO, USA), were used to treat Hepa1-6 cells for 12 h; similarly, BafA1 (100 nM, B1793, Sigma-Aldrich) and 3-MA (10 mM, M9281, Sigma-Aldrich) were used to treat GFP-LC3/HeLa cells or Hepa1-6 cells for indicated time.
For inhibitory assays, the antagonists or inhibitors, such as RU486 (30 μM, M8046, Sigma-Aldrich, St. Louis, MO, USA), were used to treat Hepa1-6 cells for 12 h; similarly, BafA1 (100 nM, B1793, Sigma-Aldrich) and 3-MA (10 mM, M9281, Sigma-Aldrich) were used to treat GFP-LC3/HeLa cells or Hepa1-6 cells for indicated time.
4
Crif1 Regulates Neuronal Oxidative Stress
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SH-SY5Y, a human neuroblastoma cell line, and HT22, an immortalized mouse hippocampal neuronal cell line, were cultured as previously described.55 (link) Cells were transfected with cDNA for Crif1 (gifted from Dr. Shong (Chungnam University, Korea)), Sp1 (hMU003598; provided from Korea Human Gene Bank, Medical Genomics Research Center, KRIBB, Korea), Sp1 K16A or Mito-DsRed using Lipofectamine and Plus reagents (Invitrogen, Carlsbad, CA, USA) and Crif1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using RNAiMax reagents (Invitrogen) for 48 h according to the manufacturer's instructions. Cells were grown to 60% confluency and treated with vehicle (dimethyl sulfoxide) or Aβ1-42 (American Peptide, Sunnyvale, CA, USA), Aβ42–1 (Bachem, Bubendorf, Switzerland) and various reagents; MG132 (10 μM; M7449, Sigma-Aldrich, St. Louis, MO, USA), 3-MA (2 mM; M9281, Sigma-Aldrich), bafilomycin (5 nM; B1793, Sigma-Aldrich), NAC (1 mM; A7250, Sigma-Aldrich), apocynin (10 μM; A10809, Sigma-Aldrich), DPI (10 μM; D2926, Sigma-Aldrich), and H2O2 (216763, Sigma-Aldrich).
5
Modulation of Cellular Pathways
6
Modulation of Cellular Pathways
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Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl2), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
7
Chloroquine and Bafilomycin A1 Modulate Cell Response
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IMR-32 and CHP-134 cells were pretreated with indicated concentration of chloroquine diphosphate salt (further abbreviated to CQ, C6628, Sigma-Aldrich) or bafilomycin A1 (further abbreviated to Baf, B1793, Sigma-Aldrich) for 1.5 h at 37 °C and subsequently treated for a given time with either the 14G2a mAb at concentration of 40 μg/ml, or MK-5108 (further abbreviated to MK, S2770, Selleckchem) at concentration of 0.1 μM, seeded, and grown at 37 °C. Additionally, PBS-treated, water-treated or DMSO-treated control cells were included. Control cells were treated with equivalent volume of the solvent of the drug.
8
Inhibitor Compounds for NOX and Autophagy
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VAS3947 (VAS), a selective inhibitor of NOX, was purchased from Calbiochem (#532336, San Diego, CA, USA); bortezomib (or PS-341) was purchased from SelleckChem (S1013, Houston, TX, USA). N-acetyl-l -cysteine (NAC), an antioxidant, bafilomycin A1 (BafA1), an inhibitor of maturation of autophagic vacuoles, and auranofin (AUR), a selective thioredoxin reductase inhibitor, were purchased from Sigma-Aldrich (A7250, B1793, and A6733, respectively, Saint Louis, MO, USA). Stock solutions (10 mM) were made using dimethylsulfoxide (DMSO) or ethanol (EtOH) as solvents. In turn, depending on the drugs, 0.01% DMSO or EtOH were used as vehicles.
9
Autophagy Monitoring with mCherry-GFP-LC3B
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For mCherry-GFP-LC3B probe, cells transfected with adenovirus were cultured for 48 hours before experiments. For BAF treatment, after incubation with EV or Met-EV for 20 hours, cells were incubated with BAF (10 nM; Sigma-Aldrich, B1793) for 4 hours at 37°C and cultured for another 3.5 hours to be lysed for Western blotting or fixed for immunofluorescence.
10
Pharmacological Modulation of Cellular Processes
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We used double-distilled water (ddH2O) to prepare stock solutions of all pharmacological agents with the exception of bafilomycin A1, which was dissolved in DMSO. The cells in control groups were treated with the equivalent volumes of ddH2O or DMSO without the addition of the pharmacological agents. The final concentrations used in treatment of cells were 2 mM, 4 mM, or 6 mM for L-glutamine (G3202, Sigma-Aldrich, St. Louis, MO), 0.5 mM or 1 mM for ascorbic acid (A4544, Sigma-Aldrich), 80 μM for zinc sulfate (Z0251, Sigma-Aldrich), 10 μM for aurintricarboxylic acid (A1895, Sigma-Aldrich), 1 μM for bafilomycin A1 (B1793, Sigma-Aldrich), 100 μM for chloroquine (C6628, Sigma-Aldrich), and 10 mM for ammonium chloride (A9434, Sigma-Aldrich).
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