HeLa “Kyoto” cells were grown in DMEM (Gibco) and LNCaP cells in RPMI, both supplemented with 10% fetal calf serum (FCS) and maintained at 37 °C, 5% CO2. SW480 cells were grown in L-15 medium (ATCC) supplemented with 10% FCS and maintained at 37 °C. For viability assays, the amount of viable cells was determined using TACS MTT Cell Proliferation Assays (Trevigen, 4890–025-K) following the manufacturer’s recommendations.
L 15 medium
Manufactured by American Type Culture Collection
Sourced in United States
L-15 medium is a cell culture medium designed for use in open-air incubators or environments with low carbon dioxide levels. It is formulated to maintain physiological pH without the need for additional carbon dioxide supplementation.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Tacs mtt cell proliferation assay, by Bio-Techne (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by American Type Culture Collection (1 mentions)
Wnt3a, by American Type Culture Collection (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Rpmi medium, by Corning (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Dulbecco s modified eagle medium dmem, by Corning (1 mentions)
Mccoy s 5a medium, by Corning (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
Mccoy s 5a medium, by American Type Culture Collection (1 mentions)
Hct116, by Biowest (1 mentions)
Caco 2, by Biowest (1 mentions)
Mccoy s 5a, by Biowest (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
11 protocols using l 15 medium
1
Cell Culture and Viability Assay
2
Culturing MDA-MB-231 Breast Cancer Cells
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The MDA-MB-231 breast adenocarcinoma cell line was obtained from ATTC and cultured in L-15 medium (ATCC) that was supplemented with 10% fetal bovine serum (FBS; GIBCO) at 37°C.
3
Culturing Basal B Subtype TNBC Cells
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The human mesenchymal TNBC cell line MDA-MB-231 was obtained from American Type Culture Collection (ATCC). MDA-MB-231 cells were characterized as basal B subtype TNBC, with mesenchymal features that allow them to migrate readily and degrades their ability to adhere and polarize [28 (link)]. MDA-MB-231 cells were maintained in L-15 medium (ATCC) containing 10% fetal bovine serum (FBS) and 100 U/mL of penicillin/streptomycin (Invitrogen) in a humidified incubator at 37°C without added CO2.
4
Differentiation of H9 hESCs to Hepatocytes
5
Culturing Human Breast and Leukemia Cells
6
Culturing Human Colon Cancer Cell Lines
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Human colon adenocarcinoma HCT-8 cells (Cat. No. CCL-244; ATCC, Manassas, VA) were cultured as previously described [26 (link),27 (link),72 ]. Human colon adenocarcinoma SW480 cells (Cat. No. CCL-228; ATCC, Manassas, VA) were cultured in L-15 Medium (Cat. No. 30–2008; ATCC) supplemented with 10% fetal bovine serum and Penicillin Streptomycin. Human colon carcinoma HCT-116 cells (Cat. No. CCL-247; ATCC, Manassas, VA) were cultured in McCoy’s 5a Medium (Cat. No. 30–2007; ATCC) supplemented with 10% fetal bovine serum and 1× antibiotic-antimycotic (Cat. No. 15240–062; Gibco). All cells were cultured at 37°C, with optimal humidity and 5% CO2.
7
Culturing Colorectal Cancer Cell Lines
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Colorectal cancer cell lines CaCo2, HCT116 and SW480 were acquired from ATCC (Manassas, VA, USA). All cells are adherent with epithelial morphology. CaCo2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Biowest, Nuaillé, France) supplemented with 20% fetal bovine serum (FBS) (GE Healthcare-Hyclone, Chicago, IL, USA); HCT116 cells were cultured in McCoy’s 5A (Biowest, Nuaillé, France) supplemented with 10% FBS and SW480 cells in L-15 medium (ATCC) with 10% FBS. A mixture of antibiotics including 100 U/mL penicillin and 100 μg/mL streptomycin (Lonza, Basel, Switzerland) was added to all cell cultures. Cells were incubated at 37 °C and 5% CO2 in a humidified incubator (HeraCell, Heraeus, Hanau, Germany).
8
Isolation and Activation of Lymphocytes
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Whole blood was diluted in 0.9 % NaCl solution in a 1:1 ratio and lymphocytes were isolated by density gradient centrifugation using lymphoprep solution (Axis-shield PoC, Oslo, Norway) upon centrifugation at 1800 × g at 20 °C for 20 min. A clear distinct layer of mononuclear cells containing lymphocytes was carefully pipetted from the tube and cultured in L-15 medium (ATCC, Borås, Sweden), supplemented with 10 % fetal bovine serum (FBS; Sigma Aldrich, Stockholm, Sweden), at 37 °C. The lymphocytes were then stimulated with heat-killed E. fecalis (CCUG, Gothenberg University, Sweden) at an optical density (OD) of 1 and incubated at 37 °C for 3 weeks (Additional file 1 ). After 3 weeks, the medium was collected and centrifuged at 3000 × g for 10 min to pellet the cells. The supernatant was filtered using 0.45 μm sterile filters (Thermofisher Scientific, Stockholm, Sweden) and centrifuged again using 100 KDa Amicon Microcon Centrifugal Filters (Millipore, Molsheim, France) at 3000 × g for 60 min. Antibodies with molecular weight over 100KDa were recovered from the filters by pipetting and were stored at −70 °C until further use.
9
Polymeric Nanoparticle Formulation Development
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PLGA
(Lactel 50:50, mol.
weight 30 000–60 000) was purchased from Durect
Corporation (Cupertino, CA). Chitosan (mol. weight 50 000–190 000)
was purchased from Sigma-Aldrich (St. Louis, MO). DOX and PTX were
ordered from LC Laboratories, Inc. (Woburn, MA, US). Coumarin-6,
fluorescein isothiocyanate, dichloromethane (DCM), polyvinyl alcohol
(PVA), and Nile red were purchased from Sigma-Aldrich, Inc. (Saint
Louis, MO). Ready-to-use dialysis tubes [molecular weight cut-off
(MWCO), 6000–8000] were purchased from Spectrum Laboratories,
Inc. (Rancho Dominguez, CA, US). 4′,6-Diamidino-2-phenylindole
(DAPI) and Alexa Fluor 350 were obtained from Life Technologies Corporation
(Grand Island, NY, USA). Cancer cell lines (MDA-MB-231 and A-549)
and related agents including trypsin/ethylenediamine tetraacetic acid
solution, F-12K medium, L-15 medium, and fetal bovine serum were purchased
from American Type Culture Collection (ATCC) (Manassas, VA, USA).
All of the other chemicals were of analytical grade.
(Lactel 50:50, mol.
weight 30 000–60 000) was purchased from Durect
Corporation (Cupertino, CA). Chitosan (mol. weight 50 000–190 000)
was purchased from Sigma-Aldrich (St. Louis, MO). DOX and PTX were
ordered from LC Laboratories, Inc. (Woburn, MA, US). Coumarin-6,
fluorescein isothiocyanate, dichloromethane (DCM), polyvinyl alcohol
(PVA), and Nile red were purchased from Sigma-Aldrich, Inc. (Saint
Louis, MO). Ready-to-use dialysis tubes [molecular weight cut-off
(MWCO), 6000–8000] were purchased from Spectrum Laboratories,
Inc. (Rancho Dominguez, CA, US). 4′,6-Diamidino-2-phenylindole
(DAPI) and Alexa Fluor 350 were obtained from Life Technologies Corporation
(Grand Island, NY, USA). Cancer cell lines (MDA-MB-231 and A-549)
and related agents including trypsin/ethylenediamine tetraacetic acid
solution, F-12K medium, L-15 medium, and fetal bovine serum were purchased
from American Type Culture Collection (ATCC) (Manassas, VA, USA).
All of the other chemicals were of analytical grade.
10
Establishment of Stable Cell Lines with PMIS Inhibitors
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To establish stable cell lines, we used PMIS inhibitor plasmids PMIS-miR-17, PMIS-miR-18a, PMIS-miR-19a, and PMIS-miR-92a-1, and the construction of these vectors has been previously described.20 (link) To produce lentivirus, 100-mm dishes of HEK293FT cells were transfected with a mix of psPAX2, pMD2.G, and the desired inhibitor construct using PEI (3:1 PEI:DNA ratio). After 48 h, cells and media containing the virus were harvested. Cells were pelleted and lysed and the filtered supernatant was used to infect ATC SW579 and MDA-T32 cells. Both cell lines were successfully transduced and expressed the PMIS inhibitor construct and GFP several days after infection. These cells were purified by fluorescence-activated cell sorting (FACS) sorting and puromycin (1 μg/mL) selection and maintained in L-15 medium according to the American Type Culture Collection (ATCC) protocol. MDA-T32 cells (ATCC, CRL3351) were cultured in RPMI with 10% FBS serum.
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