MDA-MB-231 (MDA-231) cells were purchased from the American Type Culture Collection (USA). MDA-MB-231-HM (MDA-231-HM) cells with a high potential to metastasize to the lungs were established by our institute according to a previously described method [15 (link)]. The MDA-231 gemcitabine-resistant cell subline (MDA-231-R) and MDA-231-HM gemcitabine-resistant cell subline (MDA-231-HM-R) were established according to a previous study [16 (link)] by stepwise selection with increasing concentrations of gemcitabine (Eli Lilly, USA) with 12 cycles at a range of 12–720 nmol/l in the culture medium. All of the cell lines were tested and authenticated shortly before use. Breast cancer cell lines were cultured in the American Type Culture Collection-recommended media. Cells were cultured as a monolayer in 100% air with no CO2 in a humidified incubator at 37 °C and collected during their exponential growth phase. Cells were cultured for 24 h till attachment before experimental use. All cell lines were recently authenticated by STR profiling and tested for mycoplasma contamination. GW4869 was purchased from MedChemExpress (China). Lapatinib was purchased from Absin Bioscience Inc.,(China).
Gemcitabine
Manufactured by Eli Lilly
Sourced in United States, Japan, France, Germany, United Kingdom, Spain, Canada, Australia
Gemcitabine is a synthetic nucleoside analog used as a laboratory reagent. It functions as an antimetabolite, inhibiting DNA synthesis and cell division.
Automatically generated - may contain errors
Lab products found in correlation
Bxpc 3, by American Type Culture Collection (9 mentions)
Panc 1, by American Type Culture Collection (8 mentions)
Gemzar, by Eli Lilly (7 mentions)
Mia paca 2, by American Type Culture Collection (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Paclitaxel, by Merck Group (6 mentions)
Aspc 1, by American Type Culture Collection (6 mentions)
Cfpac 1, by American Type Culture Collection (6 mentions)
Cisplatin, by Merck Group (5 mentions)
Erlotinib, by Selleck Chemicals (5 mentions)
Oxaliplatin, by Merck Group (4 mentions)
Mitomycin c, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Doxorubicin, by Merck Group (3 mentions)
Cisplatin, by Selleck Chemicals (3 mentions)
Panc 04, by American Type Culture Collection (3 mentions)
Pemetrexed, by Eli Lilly (3 mentions)
Mg132, by Merck Group (3 mentions)
Sw1990, by American Type Culture Collection (3 mentions)
Erlotinib, by Roche (3 mentions)
141 protocols using gemcitabine
1
Metastatic Breast Cancer Cell Lines
2
Establishment of Gemcitabine-Resistant Cancer Cell Lines
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To establish sequential gemcitabine-resistance cancer (GRC) cell lines, parental T24 cells were treated with 1.5 μM gemcitabine (Eli Lilly and company, IN, USA) in a culture medium for 72 h when cells reached a confluence of ~90% (Fig. 1A ). DMEM containing gemcitabine was replaced, and viability was monitored every 2–3 days. When the GRC cell lines proliferated to form colonies, they further proliferated in DMEM and were named Phase 1 (P1). The constructed P1 was further cultured to make cell stocks, and some cells were moved to a medium containing gemcitabine to build the next phase. In the same methods, the GRC cell lines from parental phase P0 to late phase P15 were constructed. A total of four types of GRC cell lines were constructed and named GRC1-4.
3
Gemcitabine Cytotoxicity Assay Protocol
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After transfected with siRNA or plasmid for 36 h, followed by further gemcitabine treatment for 12 h, cells were planted in 96 well plates with a density of 7,000 cells/well and treated with increasing amounts of gemcitabine (Eli Lilly and Co.) for 48 h or 72 h. Then, 20μL of 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma) (5 mg/ml) was added and incubated for 4 h. The supernatant was replaced with 150 μl of dimethyl sulfoxide (Sigma) and read at 490nm using a microplate photometer. Every concentration had 5 replicate wells and each group was repeated thrice.
4
Murine A20 Lymphoma Model and Combination Therapy
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BALB/c mice were inoculated subcutaneously with 2x105 A20 lymphoma cells, palpable subcutaneous tumor foci were detected under the skin within fourteen days. Large tumor blocks with a minimal volume of 700–1,000 mm3 were usually detected twenty-seven to thirty days after inoculation. The mice were then injected intraperitoneally with a single dose of 120 mg/kg of gemcitabine (Eli Lilly, Indianapolis, IN), or intratumorally with 5x105 cultured DCs after 48 hours, or injected with both gemcitabine and DCs. Control mice received saline only. On selected days after treatment, spleen cells were isolated and the single cell suspensions were subjected to flow cytometry. Tumors volumes were estimated using the formula: (π x long axis x short axis x short axis) / 6. Each control or experimental group had five to ten mice. Mice survival was observed, recorded and represented by Kaplan-Meier survival curves. Mice with certain treatments survived beyond the endpoint (120 days), and were euthanized with CO2 followed by cervical dislocation.
5
In vitro analysis of mesothelioma cell lines
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Three human MPM cell lines (H28, H2452 and MSTO-211H) were obtained from ATCC (Manassas, VA) and cultured as previously described.26 (link) Human primary DMPM cultures (MesoII and STO) were derived from tumour samples of patients who underwent surgery, and were maintained in DMEM/F12 (Gibco) under standard culture conditions for less than 20 passages.24 (link) Cells were routinely tested for mycoplasma. Analyses of mitochondrial function and glycolysis of MSTO-211H and H2452 cells were performed with the Seahorse XFp Metabolic Flux Analyzer (Agilent Technologies, Santa Clara, CA), showing that these cells have both a normal aerobic metabolism and aerobic glycolysis, though to a different extent, as reported in the Supplemental Methods and Supplementary Fig. S1 .
Co-transduction of MesoII cells with luciferase vectors and Firefly-luciferase (F-luc) activity assessment were performed according to previously established methods.23 (link),24 (link) Pemetrexed and gemcitabine were gifts from Eli Lilly (Indianapolis, IN), while the LDH-A inhibitors NHI-2 and NHI-Glc-2 were synthesised as described previously.17 (link),27 (link) Drugs were dissolved in sterile water (gemcitabine) or dimethyl sulfoxide (DMSO, Pemetrexed and LDH-A inhibitors) and diluted in culture medium immediately before use. In each experiment, we did not use concentrations higher than 0.1% DMSO.
Co-transduction of MesoII cells with luciferase vectors and Firefly-luciferase (F-luc) activity assessment were performed according to previously established methods.23 (link),24 (link) Pemetrexed and gemcitabine were gifts from Eli Lilly (Indianapolis, IN), while the LDH-A inhibitors NHI-2 and NHI-Glc-2 were synthesised as described previously.17 (link),27 (link) Drugs were dissolved in sterile water (gemcitabine) or dimethyl sulfoxide (DMSO, Pemetrexed and LDH-A inhibitors) and diluted in culture medium immediately before use. In each experiment, we did not use concentrations higher than 0.1% DMSO.
6
Synergistic Effects of Gemcitabine and VV-ING4
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SW1990 and PANC-1 cells in 96-well plates were treated with different doses of gemcitabine (µM) (Eli Lilly, Indianapolis, USA) or VV-ING4 (MOI), or combination with a constant ratio of gemcitabine/VV-ING4 of 2.5/1. Cell viability was measured 48 hrs later, based on which the synergism between gemcitabine and VV-ING4 was analyzed with Calcusyn Software (Biosoft, Cambridge, UK). For combination index plots, CI is expressed as the log10 (CI) ± 1.96 SD, the 95% confidence intervals are shown where estimable. CI values were calculated over a scope of levels of growth inhibition (GI) from 20% to 80% of the fraction affected.
7
Gemcitabine and DMBA Immune Protocol
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Gemcitabine was purchased from Eli Lilly (Indiana, USA). Granulocyte–macrophage colony-stimulating factor (GM-CSF) were purchased from BD Biosciences (New Jersey, USA). Aspirin, atorvastatine (Lipitor), carboxyfluorescein succinimidyl amino ester (CFSE), dimethylbenzanthracene (DMBA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, USA). CV-7 PTFe suture lines were purchased from GORE-TEX Suture (W.L. Gore & Associates, Inc, Arizona, USA). The antibodies were purchased from BD Biosciences (New Jersey, USA)., Biolegend (San Diego, USA) or Santa Cruz Biotechnology (Texas, USA). All of the antibodies used in this study were listed in the (Additional file 1 : Table S1).
8
Quantifying Apoptosis in MYL9-Overexpressing Cells
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To evaluate the level of apoptosis, we performed flow cytometry analysis of Annexin V, along with caspase‐3/7 activation. Annexin V assay was performed using the Annexin V‐FITC Apoptosis Detection Kit (Bio Vision, Milpitas, CA), in accordance with the manufacturer's protocol. After 72 h of each treatment, cells were harvested and stained with Annexin V‐FITC and propidium iodide (Dojindo). Caspase‐3/7 activity was evaluated using the caspase‐Glo® 3/7 Assay Kit from Promega (Madison, WI), and the relative luminescence (RLU) was measured using a GloMax® Microplate Luminometer (Promega). MYL9‐overexpressing PSN1 cells did not induce apoptosis under normal conditions; therefore, 4 nM gemcitabine (Eli Lilly Pharmaceuticals, Indianapolis) was added to the cells. The BD FACS Canto TM II system (BD Biosciences, Tokyo, Japan) was used to perform flow cytometric analysis.
9
Investigating the Role of STAT3 in EMT
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Fetal bovine serum (FBS), RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Carlsbad, CA, USA). Gemcitabine was obtained from Eli Lilly (Indianapolis, IN, USA). Antibodies against STAT3, phospho-STAT3, E-cadherin and Snail were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ALDH1A1, Ki67, and N-cadherin were purchased from Abcam (Cambridge, MA, USA). The antibody against SOCS5 was purchased from OriGene Technologies (Rockville, MD, USA). The anti-β-actin antibody used in this study was purchased from Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan). Recombinant human IL-6 was obtained from PeproTech (Rocky Hill, NJ, USA), and AG490 was purchased from Selleckchem (Houston, TX, USA).
10
Synergistic Effects of FAO Inhibitors and Gemcitabine on PDAC Cells
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We tested the sensitivity of PDAC cells to three different compounds targeting the Fatty Acid Oxidation (FAO) pathway: etomoxir, perhexiline, and trimetazidine (all provided by Sigma-Aldrich, Saint-Quentin Fallavier, France), and to the chemotherapeutic gemcitabine (Gemzar, Eli Lilly & Co). Cells were seeded in 96-well plates (5,000 cells per well) and 24 hours later, the medium was supplemented with increasing concentrations of the drugs in triplicates. For the combination treatments, we used perhexiline at 5 μM with increasing doses of gemcitabine. Viability was determined 72 hours later by the Crystal violet assay, which is independent from cell metabolism. For this, cells were fixed in glutaraldehyde (1%), washed twice with PBS, stained with Crystal violet (0.1%) for 10 min, and then washed three times with PBS. Crystals were solubilized in SDS (1%), and absorbance was measured at 600 nm using an Epoch-Biotek spectrophotometer.
We calculated the synergistic effect of the combination treatments as previously reported.28 (link) To calculate the predicted values, the cell viability of gemcitabine-treated cells was multiplied by the cell viability of perhexiline-treated cells. A synergistic effect was considered when the observed value is lower than the predicted value.
We calculated the synergistic effect of the combination treatments as previously reported.28 (link) To calculate the predicted values, the cell viability of gemcitabine-treated cells was multiplied by the cell viability of perhexiline-treated cells. A synergistic effect was considered when the observed value is lower than the predicted value.
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