The cytotoxicity of human ex vivo expanded γδ T cells on osteosarcoma cells was evaluated by MTS assay. Osteosarcoma cell lines as well as primary tumor cells were seeded in 96-well plates at 3–5 × 103 cells/well. After 24 h, they were pre-treated with VPA (Sigma) or/and ZOL at indicated concentrations for 24 h before co-cultured with γδ T cells from healthy volunteers or osteosarcoma patients at various E:T ratios. After co-culture for 2 h at 37°C, the supernatant was removed and the wells were softly washed with PBS twice. Then the cytotoxic effect was measured by MTS assay following the manufacturer’s instructions. A MR7000 microplate reader (Dynatech, NV, USA) was used to quantify the percentage of survived cells by determining the optical density. To inhibit mevalonate intermediates-mediated recognition by γδ T cells, osteosarcoma cells were treated with Mevastatin (Sigma) at 5 µM 1 h prior to treatment with VPA or/and ZOL for 24 h. Mevastatin was re-added at time of co-culture to maintain a constant concentration. To inhibit perforin-mediated cytotoxicity, γδ T cells were incubated with concanamycin A (CMA, Sigma) at 100 ng/ml for 2 h at 37°C before co-culture. To block the relevant cytotoxic pathways, specific mAbs were used at 10 mg/ml just before co-incubation assay.
Mevastatin
Manufactured by Merck Group
Sourced in United States, United Kingdom
Mevastatin is a laboratory compound used for research purposes. It is a fungal metabolite that serves as a starting point for the development of statin drugs, which are used to lower cholesterol levels in the human body. Mevastatin inhibits the activity of the enzyme HMG-CoA reductase, a key enzyme involved in the biosynthesis of cholesterol. This compound is utilized in various research settings to study the mechanisms and effects of cholesterol-lowering agents.
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Lab products found in correlation
Simvastatin, by Merck Group (9 mentions)
Lovastatin, by Merck Group (5 mentions)
Atorvastatin, by Merck Group (4 mentions)
Fluvastatin, by Merck Group (3 mentions)
Mevalonolactone, by Merck Group (3 mentions)
Mevalonic acid, by Merck Group (3 mentions)
Mevalonate, by Merck Group (2 mentions)
Gsk2033, by Bio-Techne (2 mentions)
Rosuvastatin, by Merck Group (2 mentions)
M2 10b4 cells, by American Type Culture Collection (2 mentions)
Mouse leptin quantikine elisa kit, by R&D Systems (2 mentions)
Cd 1 mice, by Charles River Laboratories (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Pravastatin, by Merck Group (2 mentions)
Mk571, by Cayman Chemical (2 mentions)
Hplc grade solvents, by Thermo Fisher Scientific (2 mentions)
Sphingosine 1 phosphate (s1p), by Merck Group (2 mentions)
Leukotriene c4, by Cayman Chemical (2 mentions)
Geranylgeranyl pyrophosphate, by Cayman Chemical (2 mentions)
Ibuprofen, by Merck Group (2 mentions)
34 protocols using mevastatin
1
Cytotoxicity of Expanded γδ T Cells on Osteosarcoma
2
Macrophage Cholesterol Homeostasis Regulation
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25-HC and mevastatin were purchased from Sigma (Sigma-Aldrich, UK). MG132 (Z-Leu-Leu-Leu-al) (CAS number: 133407-82-6) was purchased from Cambridge Bioscience. Murine recombinant IFN- was purchased from Perbio Science or Life Technology (PMC4033). Medium A : DMEM/F12 with GlutaMAX (Gibco, Life technologies) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Life technologies), 10% (v/v) L929 containing colony-stimulating factor 1 (Csf1) and Penicillin/streptomycin (PS) (Gibco, Life technologies); Medium B : DMEM/F12 with GlutaMAX supplemented with 3% (v/v) lipoprotein depleted serum (LPDS) (Sigma), 10% (v/v) L929 containing Csf1 and PS; Medium C : DMEM/F12 with GlutaMAX supplemented with 3% (v/v) LPDS, 0.01 μM mevastatin, 10% (v/v) L929 containing Csf1 and PS. The following antibodies were used: monoclonal mouse anti-HMGCR (C-1, Santa Cruz Biotechnology), rabbit anti- -actin (4967, Cell Signaling) and rabbit anti- -tubulin (ab6046, Abcam).
3
Neutrophil NET Formation Assay
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Neutrophils were seeded in a density of 2 × 105 cells per well in a 48-well suspension cell plate (Greiner bio-one) containing poly-L-lysin (0.01%, Sigma, St. Louis, MO, USA) coated glass coverslips. Cells were stimulated with either 25 nM Phorbol-12-myristat-13-acetate (PMA, Sigma), 10 mM Methyl-β-cyclodextrin (Sigma) for murine NET assays, 10 mM and 20 mM Methyl-β-cyclodextrin for human NET assays, Simvastatin 10 µM (Sigma), Mevastatin 50 µM (Sigma), or RPMI 1640 without phenol red as control, for 3 h under normoxia (18–21% O2) or hypoxia (1% O2) in a hypoxia glove box (COY Laboratories). After incubation, plates were shortly centrifuged for 5 min at 370× g, to bring down cells and NETs onto the coverslip. Finally, the cells were fixed with paraformaldehyde (PFA) at 4% final concentration for 15 min at RT and afterward stored at 4 °C until immune fluorescence staining.
4
Culturing Chlamydomonas reinhardtii for Experiments
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The C. reinhardtii wild-type strain was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection, Chinese Academy of Science, Wuhan, China). C. reinhardtii cells were cultured in liquid tris-acetate phosphate media (TAP) under 100 μmol (photons) m−2 s−1 white light with a 12 h photoperiod at 25 °C. Three-day-old cells in the logarithmic growth phase were collected for further experiments [26 (link)].
Fumagillin, mevastatin, radicicol, wortmannin, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), dimethyl sulfoxide (DMSO), and other chemical agents were purchased from Sigma-Aldrich (Shanghai, China). Four mycotoxins and DCMU stock solutions were dissolved in 100% DMSO and further diluted with sterile water. The final concentration of DMSO in the working solutions of all chemical agents was less than 1% (v/v).
Fumagillin, mevastatin, radicicol, wortmannin, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), dimethyl sulfoxide (DMSO), and other chemical agents were purchased from Sigma-Aldrich (Shanghai, China). Four mycotoxins and DCMU stock solutions were dissolved in 100% DMSO and further diluted with sterile water. The final concentration of DMSO in the working solutions of all chemical agents was less than 1% (v/v).
5
Neural stem cell lipid modulation
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NSCs were plated at the density of 1×105 cells/mL on 2% Matrigel coated surface. Cells were allowed to proliferate and reach 80%–90% confluency before treatments were carried out. Overnight treatments (16 hours) were applied unless indicated otherwise. Twelve percent fatty acid–free BSA (Sigma-Aldrich, St. Louis, MO) was used as carriers for all lipids used for cell culture treatment. All control wells were treated with 12% BSA. Other compounds and final concentration used in this study are as follows: T0901317 (1 μM, Sigma-Aldrich, St. Louis, MO), GSK2033 (10 μM, Tocris, Bristol, UK), Mevastatin (50 μM, Sigma-Aldrich, St. Louis, MO), mevalonate (50 μM, Sigma-Aldrich, St. Louis, MO), 25-HC (1 μg/mL, Sigma-Aldrich, St. Louis, MO), and Cholesterol (10 μg/mL, Sigma-Aldrich, St. Louis, MO).
6
Prostate Cancer Cell Lines and Stromal Cells
7
Cholesterol Depletion Alters Claudin Localization
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COS-7 cells were transfected with pSNAP-Cldn2 and pYFP-Cldn10a with a total plasmid conc. of 1.6 µg/ml and treated with 10 µM Mevastatin (Sigma-Aldrich, #567022-5MG) for 24 h in DMEM with FBS or 10 mM Methyl-β-cyclo-dextrin (MβCD) (Sigma-Aldrich, #C4555-1G) for 1 h in DMEM without FBS. As control in an additional approach the equal amount of H2O was used. The transfected cells were labeled with BG-Atto590 and immunofluorescent stained with α-GFP-NB-Atto647N (1:200) and imaged in two-color STED.
For Filipin III staining, the cells were fixed with 4% PFA/Sucrose for 15 min, quenched with 0.1 M glycine in PBS+ for 30 min and incubated for 2 h with a freshly prepared Filipin III solution (final concentration: 0.05 µg/ml) (Sigma-Aldrich, #F4767-1MG) under light protection. The cells were washed with PBS+ and imaged with a Leica SP8 TCS microscope (Leica Microsystems). Filipin III staining was imaged with the following settings: for excitation, a UV-laser was used (405 nm) and the emission filter was set to 415–470 nm. The fluorescence signal was detected by a photomultiplier tube (PMT). Images were acquired with a HC PL APO CS2 100 × /1.40 NA oil objective (Leica Microsystems), a scanning format of 1024 × 1024 pixels, 8-bit sampling, 6x line averaging and 1x optical zoom, yielding a pixel dimension of 113 × 113 nm.
For Filipin III staining, the cells were fixed with 4% PFA/Sucrose for 15 min, quenched with 0.1 M glycine in PBS+ for 30 min and incubated for 2 h with a freshly prepared Filipin III solution (final concentration: 0.05 µg/ml) (Sigma-Aldrich, #F4767-1MG) under light protection. The cells were washed with PBS+ and imaged with a Leica SP8 TCS microscope (Leica Microsystems). Filipin III staining was imaged with the following settings: for excitation, a UV-laser was used (405 nm) and the emission filter was set to 415–470 nm. The fluorescence signal was detected by a photomultiplier tube (PMT). Images were acquired with a HC PL APO CS2 100 × /1.40 NA oil objective (Leica Microsystems), a scanning format of 1024 × 1024 pixels, 8-bit sampling, 6x line averaging and 1x optical zoom, yielding a pixel dimension of 113 × 113 nm.
8
Interleukin-2 and Interleukin-15 Cytokine Protocol
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Recombinant human interleukin (IL)-2 and IL-15 were purchased from Nipro (Osaka, Japan) and PeproTech Inc., (Rocky Hill, NJ, USA). Zol (Zometa) was purchased from Novartis (Basel, Switzerland). Mevastatin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Mevastatin-Induced Cellular Responses
10
Comprehensive Glioma Signaling Pathway Analysis
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Smad3 (phos-S208) (Abcam, ab138659), SERPINE1 (Fisher Scientific), Rap 1 (sc-398755) and TGF-β RI Kinase inhibitor V (Santa Cruz Biotechnology), PARP (#9542S), LC3 A/B (#4108S), ZYX, and total Smad3 (Cell Signaling Technology) antibodies were obtained from commercial vendors. Simvastatin was obtained from Sigma Chemical (in vitro stock and in vivo stock for subcutaneous GIC grafts) and Selleckchem (in vivo stock for GIC intracranially grafted mice). TGF-β2, mevastatin, fluvastatin, lovastatin, geranylgeranyl pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) and (±)-mevalonolactone were obtained from Sigma Chemical. Silencer® select pre-designed SMAD3 siRNAs, and Silencer® Select Negative Control siRNA were purchased from Life Technology. LY2109761 was from AdooQ BioScience (A11133).
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