All cell lines (DLD1, LoVo, HCT116, HT-29, HCT-8, SW480, SW620, and T84) except HCEC were obtained from the ATCC and were minimally passaged. Human colonic epithelial cells (HCEC) were immortalized by expression of cyclin dependent kinase 4 (Cdk4) and the active components of human telomerase (hTERT) and were kindly provided by Dr. Jerry Shay (UT Southwestern, Dallas TX). HCEC cells were maintained in DMEM media with 2% calf serum, 25 ng/ml EGF (Sigma Aldrich, St. Louis, MO), 2 μg/ml transferrin (Sigma Aldrich, St. Louis, MO), 10 μg/ml insulin (Sigma Aldrich), 5 nM sodium selenite (Sigma Aldrich, St. Louis, MO), 1 μg/ml hydrocortisone (Sigma Aldrich), and 50 μg/ml gentamicin (Gemini Bio-products, West Sacramento, CA). HCEC cells were grown at 37 °C on Primaria flasks (Corning Inc., Conring NY) placed in chambers purged with 93% nitrogen 5% carbon dioxide and 2% oxygen gas. All other cell lines were maintained in DMEM with 4 mM L-glutamine, 10% FBS, and 1x penicillin/streptomycin at 37 °C and 5% carbon dioxide.
Primaria flasks
Manufactured by Corning
Sourced in United States
Primaria flasks are laboratory equipment designed for cell and tissue culture applications. They feature a treated surface that promotes cell attachment and growth. These flasks are available in various sizes to accommodate different volume requirements.
Automatically generated - may contain errors
Lab products found in correlation
Hydrocortisone, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Sodium selenite, by Merck Group (1 mentions)
Gentamicin, by Gemini Bio (1 mentions)
Laminin, by Merck Group (1 mentions)
Neurocult ns a basal medium, by STEMCELL (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
4 protocols using primaria flasks
1
Colonic Epithelial Cell Culture Conditions
2
Glioma Neural Stem Cell Culture Protocol
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Patient-derived glioma neural stem cells (GNS 411) were a gift from the laboratory of P. Dirks, with Research Ethics Board approval at the Hospital for Sick Children, Toronto and the University of Toronto. The characterization of these cells as GSC-enriched cultures is presented in several publications by Park et al. and Dolma et al. (45 (link), 46 (link)). These cells are referred to as GSCs throughout the manuscript. Cells were maintained in an incubator (37°C, 5% CO2, and 95% humidity) grown in Corning Primaria flasks (Corning, 353808) coated with poly-l -ornithine (PLO; Sigma-Aldrich, P4957) and laminin (Sigma-Aldrich, 11243217001). GSC growth media contained serum-free NeuroCult NS-A Basal Medium (STEMCELL Technologies, 05750) supplemented with N2, B27, EGF, fibroblast growth factor, and heparin as previously described for neural stem cells (47 (link)).
3
Derivation and Culture of HGS Cell Lines
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HGS lines were derived from individual tumors collected in cold PBS, minced with a scalpel, and incubated with 1 mg/ml collagenase type I (Fisher, 17018-029). After being split once onto collagen-coated plates, cells were transferred into Primaria flasks (Corning) and then propagated as polyclonal lines on normal cell-culture plastic (Corning). Complete medium was DMEM:F12 1:1 with GlutaMax (GIBCO) with the addition of 4% FBS (Hyclone), 1x Pen/Strep (Invitrogen), 1x insulin/transferrin/selenium (Invitrogen, 51300), 100 ng/ml hydrocortisone (Sigma, H0135), 20 ng/ml murine Epidermal Growth Factor (EGF, Sigma, E4127), 1x antibiotic-antimycotic (GIBCO). Cells were trypsinized with 0.05% trypsin-EDTA (GIBCO). 60577 and 30200 cell lines were derived and cultured as described (Szabova et al., 2014 (link)).
4
Isolation and Characterization of Rat Endometrial Epithelial Cells
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Considering the differences among human samples, primary rEECs were collected from the endometrium of 8-week-old female Wistar rats (weight 200–250 g, Southern Medical University Animal Experiment Center) to maintain consistency. Eligible Wistar rats were anesthetized with intraperitoneal injection of 4% chloralic hydrate (Hatch, Shanghai, China). A laparotomy was performed on the ventral midline and endometrium specimens were scraped from incised uterus. Specimens were rinsed and minced into small pieces, digested and separated according to the methods of Ngô et al. (2009) (link). rEECs were retained on 40 μm aperture sieves and plated onto Primaria flasks (Corning, USA). Dulbecco's modified Eagle's medium (DMEM) (Gibco Invitrogen, France) with 10% fetal calf serum (Gibco Invitrogen, France) were applied to culture cells. Purification of rEECs were confirmed by staining with a 1:1000 dilution of fluorescein isothiocyanate labeled anti-cytokeratin and anti-vimentin antibodies (Cell Signaling Technology, USA). The Olympus fluorescent microscope (Germany) was used to analyze fluorescence and capture pictures.
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