Cambinol (Sigma) was resuspended in DMSO to a stock concentration of 1 mM. For the Cambinol experiments, cells were first infected with purified JCPyV in serum-free media (SFM). After 2 hr virus inoculum was removed and cells were washed with 1X PBS, then complete EV-depleted media with 10 μM Cambinol or a volume-matched vehicle control was added. At 6 days post infection (dpi), additional media with drug or vehicle control was added to cells at the same final concentration (96w format – 50 μL; 24w format – 0.5 mL).
Cambinol
Manufactured by Merck Group
Sourced in United States
Cambinol is a laboratory instrument used for the detection and quantification of chemical compounds. It utilizes advanced spectroscopic techniques to analyze the composition and properties of various samples. The core function of Cambinol is to provide accurate and reliable data to support research and analytical activities in a wide range of scientific disciplines.
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Lab products found in correlation
Nicotinamide, by Merck Group (3 mentions)
Ex 527, by Merck Group (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Rpmi8226, by American Type Culture Collection (1 mentions)
Cell counting kit 8 assay, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Fluorescence activated cell sorter, by BD (1 mentions)
Protein marker, by Thermo Fisher Scientific (1 mentions)
Dna ladder, by New England Biolabs (1 mentions)
Ni2 nta agarose, by Qiagen (1 mentions)
Amylose resin, by Qiagen (1 mentions)
Sirtinol, by Merck Group (1 mentions)
Dna modifying enzymes, by New England Biolabs (1 mentions)
Sirt1 fluorometric drug discovery kit, by Enzo Life Sciences (1 mentions)
Altenusin, by Enzo Life Sciences (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Phalloidin 488, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
15 protocols using cambinol
1
Cambinol Inhibits JCPyV Infection
2
Cambinol Inhibits JCPyV Infection
3
Apoptosis Induction in Myeloma Cells
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MM cell lines RPMI8226 and U266 were ordered from the American Type Culture Collection. A Cell Counting Kit (CCK)-8 assay was purchased from Beyotime Institute of Biotechnology. Cambinol, Annexin V and PI were purchased from Sigma-Aldrich (Merck KGaA). Anti-procaspase-3 (cat. no. 610322; 1:1,000), and poly(ADP-ribose) polymerase 1 (PARP) (cat. no. 556362; 1:2,000) antibodies were purchased from BD Biosciences. The anti-β-actin antibody (cat. no. ZRB1312; 1:1,000) was purchased from Sigma-Aldrich (Merck KGaA). Anti-p53 (cat. no. 48818; 1:1,000), cleaved caspase 3 (cat. no. 9664; 1:1,000), acetylated p53 (Lys382; Ac-p53) (cat. no.2525; 1:1,000), Bcl-2 (cat. no. 15071; 1:1,000), cyclin D1 (cat. no.2922; 1:1,000) and p21 (cat. no. 2947; 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. The anti-mouse and rabbit horseradish peroxidase-conjugated secondary antibodies (cat. no. 7076, 7074; 1:5,000) were purchased from Cell Signaling Technology, Inc. Culture RPMI-1640 medium and FBS were purchased from Cytiva. Fluorescence Activated Cell Sorter (FACS) was obtained from BD Biosciences.
4
Purification and Biochemical Characterization of Sirtuins
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All restriction enzymes, DNA-modifying enzymes, and DNA ladders were obtained from New England Biolabs. Plasmid pETM-41 and TEV protease were kindly provided by Dr Amit Sharma (ICGEB, New Delhi). Protein markers were obtained from Thermo Fisher Scientific (USA). nicotinamide Adenine Dinucleotide [Adenylate-32P] (800Ci/mmol) was purchased from American Radiolabeled Chemicals (USA). Ni2+-NTA agarose and amylose resin were purchased from Qiagen and New England Biolabs, respectively. Sirtinol, nicotinamide, cambinol, and Ex-527 were obtained from Sigma-Aldrich (USA). SIRT1 Fluorometric Drug Discovery Kit and SIRT5 Fluorometric Drug Discovery Kit were procured from Enzo Life Sciences (USA). Other materials used in this study were of analytical grade and were commercially available.
5
Inhibiting nSMase2 with Cambinol
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Cambinol [5-(2-hydroxynaphthalen-1-ylmethyl)-6-phenyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one; Sigma-Aldrich], a pharmaceutical inhibitor of nSMase2 (Figuera-Losada et al., 2015 (link)), was prepared at a stock concentration of 20 mM in DMSO and added to donor cells at a final concentration of 10 μM for 48 h in serum-free culture media.
6
Pretreatment with nSMase2 Inhibitors
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7
Multimodal Analysis of Autophagy Regulation
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Cambinol, Actinomycin D, TSA, Rapamycin, and Nicotinamide were purchased from Sigma (St. Louis, MO). AK1 and AGK2 were purchased from ChemBridge (San Diego, Ca). EX527 was supplied by Selleck Chemicals (Houston, TX). UO126 was purchased from Calbiochem (Billerica, MA) and used at 10 μM. LAMP-1 antibody (h4A3) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa and used at a 1:200 dilution. GM130 and EEA1 antibodies (BD Biosciences, San Jose, CA) were used at 1:50. Mitotracker Red (Molecular Probes, Carlsbad, CA) was used according to the manufacturer's protocol. The SIRT1 (B-10) and Ac-tubulin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX) and was used at 1:1000. pMEK1/2 S271/221, p70 S6 Kinase Thr389, pErk1/2 T202/Y204, Ac-p53 Lys382, LC3I/II, and total Erk1/2 antibodies (Cell Signaling Technology, Beverly, MA) were used at 1:1000 for western blot. Anti-tubulin antibody (NeoMarkers, Fremont, CA) was used at 1:100 for immunofluorescence (IF) and 1:20,000 for western blot analysis. Secondary antibodies include Dylight 594 donkey anti-mouse 1:100 (Jackson IR, West Grove, PA) for immunoflorescence, and HRP-conjugated anti-mouse and anti-rabbit for western blot (GE Healthcare, Pittsburgh, PA) used at 1:5000. Phalloidin 488 (Invitrogen, Carlsbad, CA) was used at 1:200.
8
Preparation of CAM and CDDP Stock Solutions
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Cambinol (CAM) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of CAM (100 mM) was prepared in dimethyl sulfoxide (DMSO). Cisplatin (CDDP) was purchased from Sigma (St. Louis, MO, USA) and dissolved in phosphate buffered saline (PBS) without Mg2+ and Ca2+ at 1 mg/mL as a stock solution. The drugs were diluted with culture medium to the respective concentration just before use.
9
Lipid Compound Acquisition for Experiments
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Englerin A was purchased from Ceriliant Corporation and Cfm Oskar Tropitzsch. C8- and C16-ceramides and C8-ceramide-1-phosphate were obtained from Avanti Polar Lipids. Imipramine hydrochloride and Cambinol were purchased from Sigma and Cayman Chemical, respectively.
10
Cambinol Cytotoxicity Assay in SVG-A Cells
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Cytotoxicity of cambinol (Sigma) was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) according to manufacturer’s protocols. Briefly, SVG-A cells were plated in complete media. The next day, cells were treated with cambinol diluted to several concentrations in complete media, or a volume-matched vehicle control at the highest concentration. At 3, 6, or 9 days post drug treatment, MTS reagent was added for 1–4 hrs and absorbance read at 450 nm using a Glomax Multi Detection System (Promega) plate reader. At 6 days post infection (dpi), additional media with drug or vehicle control was added to cells at the same final concentration
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