Ls180

Manufactured by European Collection of Cell Cultures

Sourced in United Kingdom, Japan

The LS180 is a laboratory equipment product. It is designed for cell culture applications. The LS180 provides temperature control and suitable environmental conditions for cell growth and maintenance.

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18 protocols using ls180

Culturing Colon Cancer Cell Lines

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The human colon adenocarcinoma cell lines, LS180 and Caco-2, were purchased from European Collection of Cell Cultures (ECACC) collections (KAC, Hyogo, Japan). LS180 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 1,500 mg/L glucose (FUJIFILM Wako Pure Chemical, Osaka, Japan) and Caco-2 cells were cultured in DMEM with 4,500 mg/L glucose. In both the cases, the medium was supplemented with heat-inactivated 10% fetal bovine serum (FBS) (Cosmo Bio, Tokyo, Japan). The cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂.
The acidic cell culture medium (pH 6.5) was obtained by the addition of 35–37% HCl solution to DMEM containing 1,500 mg/L glucose supplemented with heat-inactivated 10% FBS by measuring pH with a FiveEasy Plus FEP20 pH Meter (METTLER TOLEDO, Tokyo, Japan). LS180 cells were grown for a sufficient time in normal medium and were subsequently maintained in the acidic cell culture medium (pH 6.5) for a few days as described previously with some modifications [26 (link)–28 (link)].

Kobori T., Tameishi M., Tanaka C., Urashima Y, & Obata T. (2021). Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells. PLoS ONE, 16(5), e0250889.

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Culturing Human CRC Cell Lines

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Human CRC cell lines LS180, COLO205 and HT29, purchased from the European Collection of Cell Cultures, were maintained in RPMI 1640 (GIBCO) or, for HT29, McCoy's 5A medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, GlutaMAX-I and kanamycin (GIBCO). HMEC cells (provided by Professor T Resink, University of Basel) were cultured in EBM-2 medium (Lonza). Tumour-associated stromal cells (TASC) were expanded from CRC samples in α-MEM (GIBCO) supplemented with 10% fetal bovine serum and 5 ng/mL FGF-2 (R&D Systems).

Amicarella F., Muraro M.G., Hirt C., Cremonesi E., Padovan E., Mele V., Governa V., Han J., Huber X., Droeser R.A., Zuber M., Adamina M., Bolli M., Rosso R., Lugli A., Zlobec I., Terracciano L., Tornillo L., Zajac P., Eppenberger-Castori S., Trapani F., Oertli D, & Iezzi G. (2015). Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer. Gut, 66(4), 692-704.

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Receptor-Mediated Reporter Assays

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The following cell lines, endogenously expressing the respective receptors and stably transfected with corresponding luciferase reporter genes were used to estimate specific receptor activation: AZ-AHR cell line was used to quantify the AhR- and anti-AhR-mediated activity (Novotná et al., 2011 (link)); AZ-GR cell line was used to determine the GR-mediated activity (Novotná et al., 2012 (link)); IZ-VDRE and IZ-CYP24 cell lines were used for detection of VDR activation (Bartoňková et al., 2016 (link)); PZ-TR cell line was used for determination of TR-mediated activity (Illés et al., 2015 (link)); T47D.luc cell line, kindly provided by BioDetection Systems BV (BDS, Amsterdam, The Netherlands), was used to estimate the ER-mediated activity (Legler et al., 1999 (link)); AIZ-AR cell line was used for determination of androgen receptor (AR) activation (Bartoňková et al., 2015 (link)); HT-29 cell stably transfected with PPARγ reporter were used for determination of PPARγ-mediated activity (Tylichová et al., 2017 (link)). For detection of PXR-mediated activity, we employed human colon adenocarcinoma LS180 (purchased from the European Collection of Cell Cultures, Salisbury, UK) transiently transfected with PXR-regulated reporter gene, as described previously (Kubešová et al., 2016 (link)). Description of all individual assays is provided in Supplemental Experimental Procedures.

Pěnčíková K., Svržková L., Strapáčová S., Neča J., Bartoňková I., Dvořák Z., Hýžd’alová M., Pivnička J., Pálková L., Lehmler H.J., Li X., Vondráček J, & Machala M. (2018). In vitro profiling of toxic effects of prominent environmental lower-chlorinated PCB congeners linked with endocrine disruption and tumor promotion. Environmental pollution (Barking, Essex : 1987), 237, 473-486.

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Human Cell Lines Culture Protocol

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Experiments were carried out on human colon adenocarcinoma cell lines HT-29 and LS180, human colon epithelial cell line CCD 841 CoTr and human skin fibroblasts HSF. HT-29, LS180 and CCD 841 CoTr cell lines were obtained from the European Collection of Cell Cultures (Centre for Applied Microbiology and Research, Salisbury, UK). HSF were obtained with the outgrowth technique from skin explants from young volunteers in the Department of Virology and Immunology, UMCS, Lublin (Poland).
HT-29, LS180, CCD 841 CoTr and HSF cells were cultured in 1:1 mixture of DMEM and Nutrient mixture F-12 Ham. Media were supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL). The medium was changed every two days. The cells were grown in 25 cm² flasks (Nunc, Roskilde, Denmark) and kept in a humidified atmosphere with 5% CO₂ at 37 °C or 33 °C (CCD 841 CoTr).

Czerwonka A., Lemieszek M.K., Karpińska M., Matysiak J., Niewiadomy A, & Rzeski W. (2018). Evaluation of the effect of 2-(2,4-dihydroxyphenyl)-4H-benzofuro[3,2-d][1,3]thiazin-4-one on colon cells and its anticancer potential. Medicinal Chemistry Research, 27(9), 2150-2159.

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Culturing Colon Cancer and Normal Cells

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For the cell culture study, human colon adenocarcinoma cell line LS180 and human colonic epithelial cell line CCD841 CoN were purchased from the European Collection of Cell Cultures (ECACC, Centre for Applied Microbiology and Research, Salisbury, UK) and American Type Culture Collection (ATCC, Menassas, VA, USA), respectively. LS180 cells and CCD841 CoN cells were maintained in Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. Then, 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 g/mL) were added to the cell culture media. Cells were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

Augustynowicz D., Lemieszek M.K., Strawa J.W., Wiater A, & Tomczyk M. (2023). Phytochemical Profiling of Extracts from Rare Potentilla Species and Evaluation of Their Anticancer Potential. International Journal of Molecular Sciences, 24(5), 4836.

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Established Colon Cancer Cell Lines

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Established human colon cancer cell lines (LS180, HCT116, and HT29) were purchased from European Collection of Cell Cultures (ECACC) and immediately stored in liquid nitrogen. Cells used for specific investigations were recovered from cryopreserved aliquots and cultured for a maximum of 10 passages. LS180 and HCT116 were maintained in RPMI-1640 supplemented with 10% FBS, GlutaMAX-I, nonessential amino acids (NEAA), 100 mM sodium pyruvate, 10 mM HEPES (all from GIBCO), and 50 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), whereas HT29 was maintained in McCoy’s 5A medium (Sigma) supplemented with 10% FBS and GlutaMAX-I.
Absence of mycoplasma contamination in cultured cells was verified by PCR testing prior to investigation. In specific experiments, LS180 cells co-expressing green fluorescent protein (GFP) and firefly luciferase (GFP/Luc-LS180 cells) [27 (link)] were also used.

Mele V., Basso C., Governa V., Glaus Garzon J.F., Muraro M.G., Däster S., Nebiker C.A., Mechera R., Bolli M., Schmidt A., Geiger R., Spagnoli G.C., Christoforidis D., Majno P.E., Borsig L, & Iezzi G. (2022). Identification of TPM2 and CNN1 as Novel Prognostic Markers in Functionally Characterized Human Colon Cancer-Associated Stromal Cells. Cancers, 14(8), 2024.

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Culturing Human Colorectal Cancer Cell Lines

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Established human CRC cell lines (HCT116, LS180, COLO205, HT29 and SW480) were purchased from European Collection of Cell Cultures (ECACC, Salisbury, UK). HCT116, LS180 and COLO205 were maintained in RPMI-1640 supplemented with 10% FBS, GlutaMAX-I, non-essential amino acids (NEAA), 100 mM sodium pyruvate, 10 mM HEPES (all from GIBCO) and 50 mM 2-mercaptoethanol (Sigma–Aldrich, St. Louis, MO). HT29 was maintained in McCoy’s 5A medium (Sigma) supplemented with 10% FBS and GlutaMAX-I. SW480 were cultured in L-15 Medium (Leibovitz) (Sigma–Aldrich) supplemented with 10% FBS and GlutaMAX-I. Kanamycin sulfate (GIBCO) was included with all media. Absence of mycoplasma contamination in cultured cells was verified by PCR testing prior to investigation.

Mele V., Muraro M.G., Calabrese D., Pfaff D., Amatruda N., Amicarella F., Kvinlaug B., Bocelli-Tyndall C., Martin I., Resink T.J., Heberer M., Oertli D., Terracciano L., Spagnoli G.C, & Iezzi G. (2014). Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β. International Journal of Cancer. Journal International du Cancer, 134(11), 2583-2594.

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Overexpression of c-Src in Colon Cancer Cells

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COLO-205, SW48, SW480, LS180, LS174-T, HT-29, T84 and LoVo cells were purchased from the European Collection of Cell Cultures (ECACC). Cells in culture were maintained as a subconfluent monolayer in Dulbecco's Modified Eagle's medium supplied with non-essential amino acids (LS180 and LS174-T), Dulbecco's modified Eagle's medium-F12 nutrient mixture (T84), McCoy 5-A medium (HT-29), L15 Leivobitz medium (SW48 and SW480), Nutrient Mixture F12 HAM (LoVo) and RPMI 1640 (COLO-205) purchased from Sigma. Each cell line was grown under identical conditions, and cell culture medium supplements were provided according to the manufacturer's instructions. To ectopically overexpress c-Src kinase protein, subconfluent SW480 and T84 cells were transfected with 0.4 μg of pBABE-puro expression plasmid carrying cDNA from a c-Src gene. Stable clones of the T84 cell line were selected in F12/DMEM medium supplied with 0.5 μg of puromycin for 3 weeks. In the same way, stable clones of the SW480 cell line were selected in L15 Leivobitz medium supplied with 0.5 μg of puromycin. As a control, subconfluent SW480 and T84 cells were transfected with 0.4 μg of DNA containing an empty pBABE-puro expression plasmid. Positive clones were selected for protein expression measured by Western blotting. The entire set of transfected clones was used as a stable pool for transfection.

Perez M., Lucena-Cacace A., Marín-Gómez L.M., Padillo-Ruiz J., Robles-Frias M.J., Saez C., Garcia-Carbonero R, & Carnero A. (2016). Dasatinib, a Src inhibitor, sensitizes liver metastatic colorectal carcinoma to oxaliplatin in tumors with high levels of phospho-Src. Oncotarget, 7(22), 33111-33124.

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Proliferation Assays in LS180 Cells

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The human colon adenocarcinoma cell line LS180 was obtained from the European Collection of Cell Cultures (ECACC, Centre for Applied Microbiology and Research, Salisbury, UK). LS180 cells were grown in a 1:1 mixture of DMEM (Sigma-Aldrich, St. Louis, MO, USA) and Ham’s F-12 nutrient mixture (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich), penicilin (100 U/mL) (Sigma-Aldrich) and streptomycin (100 μg/mL) (Sigma-Aldrich). Cells were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.
The effects of extracts on cancer cells proliferation were determined by the MTT metabolism assay and BrdU incorporation immunoassay. The BrdU test was conducted using Cell Proliferation ELISA BrdU according to the manufacturer’s instructions (Roche Diagnostics GmbH, Penzberg, Germany). Both assays were performed in human adenocarcinoma cells (LS180). Details of the procedures are described elsewhere [52 (link)].

Pietrzak W., Nowak R., Gawlik-Dziki U., Lemieszek M.K, & Rzeski W. (2017). LC-ESI-MS/MS Identification of Biologically Active Phenolic Compounds in Mistletoe Berry Extracts from Different Host Trees. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(4), 624.

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Caucasian Colon Adenocarcinoma Cell Lines

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Caucasian colon adenocarcinoma cell line LS180 was purchased from European Collection of Cell Cultures (ECACC No. 87021202). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% charcoal-stripped fetal bovine serum, as recommended by supplier. LS174T cells (ATCC: CL-188; 7 000 3535) (Synthego Corporation, Redwood City, CA, USA) were the parental cells used to generate PXR knockout variant LS174-PXR-KO, using CRISPR Cas9 technology, as we described and validated recently [21 ]. Stably transfected reporter gene cell line AZ-AHR was as described elsewhere [22 (link)]. Primary human hepatocyte cultures were of two origins: (i) Long-term primary human hepatocytes in monolayer batch Hep2201016 (male, 66 years, Caucasian), Hep2201020 (male, 75 years, Caucasian), Hep2201029 (male, 89 years), Hep2201032 (male, 43 years, Caucasian) were purchased from Biopredic International (Rennes, France). (ii) Primary human hepatocytes from multiorgan donor LH79 (male, 60 years, Caucasian) were prepared at Palacky University Olomouc. Liver tissue was obtained from Faculty Hospital Olomouc, Czech Republic, and the tissue acquisition protocol followed the requirements issued by “Ethical Committee of the Faculty Hospital Olomouc, Czech Republic” and Transplantation law #285/2002 Coll.

Li H., Illés P., Karunaratne C.V., Noerdstrom L.U., Liu X., Yang A., Qiu Y., Kurland I.J., Lukin D.J., Chen W., Jiskrová E., Krasulová K., Pečinková P., DesMarais V.M., Liu Q., Albanese J.M., Akki A., Longo M., Coffin B., Dou V., Mani S, & Dvořák Z. (2021). Deciphering structural bases of intestinal and hepatic selectivity in targeting pregnane X receptor with indole-based microbial mimics. Bioorganic chemistry, 109, 104661.

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