Telomerase-immortalized human corneal epithelial cells (hTCEpi) were obtained from Dr. Jim Jester (UC Irvine), Dr. Vijay Krishna Raghunathan and Dr. Christopher J. Murphy (UC Davis).34 (link) hTCEpi cells were routinely cultured in the EpiLife® medium supplemented with an EpiLife defined growth supplement (EDGS, Life Technologies, Grand Island, USA) and 1% (v/v) penicillin/streptomycin (Life Technologies). Cells were incubated at 37 °C with 5% CO2 until they reach ~70% confluence and were used between passages 40 and 70 for all electrotaxis experiments.
Epilife defined growth supplement edgs
Manufactured by Thermo Fisher Scientific
Sourced in United States
EpiLife Defined Growth Supplement (EDGS®) is a serum-free, defined cell culture supplement designed to support the growth and proliferation of human epidermal keratinocytes in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Epilife medium, by Thermo Fisher Scientific (4 mentions)
Epilife, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin amphotericin b psf, by Lonza (2 mentions)
Supplementmix, by PromoCell (2 mentions)
Dispase, by Boehringer Ingelheim (1 mentions)
Trypsin solution, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
T25 flask, by Corning (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Collagen coated plates, by Thermo Fisher Scientific (1 mentions)
9 protocols using epilife defined growth supplement edgs
1
Culturing Telomerase-Immortalized Human Corneal Epithelial Cells
2
Establishing Primary Corneal Epithelial Cultures
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All work with human tissue was done in accordance with the tenets of Helsinki. Donor human corneoscleral rims with no history of corneal diseases that were unsuitable for transplant were used to establish primary epithelial cultures as previously described [9] (link), [38] (link). Human cadaver corneas were graciously donated by the Lions Eye Bank of Wisconsin, Madison or the Missouri Lions Eye Bank (Columbia, MO). These tissue samples obtained were not human subjects research and as such approval from committee or kin were not necessary. Briefly, sclera and limbal regions of the cornea were trimmed and the tissue was immersed in dispase (1.2 U/ml, Boehringer, Mannheim, Germany) at 37°C for 4 h. Corneal epithelial cells were removed by gently rubbing the anterior surface with a sterile pipette tip. Cell suspensions were pooled, centrifuged and re-suspended in EpiLife medium (Life Technologies, Carlsbad, CA) supplemented with EpiLife defined growth supplement (EDGS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies) and used between passages 2 and 3. Experiments were repeated with cells isolated from three different donors.
Immortalized human corneal epithelial cells (hTCEpi; [44] (link)), kindly provided by Dr James V Jester (UC Irvine), were maintained in EpiLife medium as above and were used between passages 40–60.
Immortalized human corneal epithelial cells (hTCEpi; [44] (link)), kindly provided by Dr James V Jester (UC Irvine), were maintained in EpiLife medium as above and were used between passages 40–60.
3
Cultivation of Immortalized Corneal Epithelial Cells
4
Immortalized Corneal Epithelial Cell Culture
5
Immortalized Corneal Epithelial Cell Culture
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Human telomerase reverse transcriptase-immortalized corneal epithelial (hTCEpi) cells graciously donated by James Jester, PhD (University of California Irvine), were used between passage 36 and 50. The hTCEpi cells were cultured in growth medium composed of EpiLife® (Life Technologies; Carlsbad, CA) supplemented with 1% EpiLife Defined Growth Supplement (EDGS®; a proprietary combination of bovine serum albumin, bovine transferrin, hydrocortisone, recombinant human-like growth factor type-1, prostaglandin, and recombinant human epidermal growth factor (Life Technologies) and 1% penicillin-streptomycin-amphotericin B (PSF, Lonza; Walkersville, MD).
6
Isolation and Culture of Oral Keratinocytes
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As described previously, with modification, 12 mucosal tissue was digested overnight with 0.04% trypsin solution (Life Technologies, Carlsbad, CA) with 19.25 mg/mL of gentamicin (Life Technologies) and 0.765 mg/mL of fungizone (Life Technologies) at room temperature and transferred into 0.0125% defined trypsin inhibitor (DTI; Life Technologies) to neutralize the trypsin solution. Oral keratinocytes were mechanically dissociated from the submucosal connective tissue, collected, and centrifuged. Dissociated oral keratinocytes were resuspended in a chemically defined culture system (EpiLife Ò , supplemented with Epilife defined growth supplement (EDGS) and 0.06 mM calcium; Life Technologies) and seeded into one T-25 flask (Corning, Corning, NY). Ambient oxygen in an incubator at 37°C in a humidified 5.0% CO 2 environment was used for following the culture procedure. For the serial cultures, cells were de-tached in 0.025% trypsin/EDTA (Life Technologies) and neutralized by DTI. P2-P4 were used for the experiments.
7
Isolation and Culture of Human Hair Follicle Cells
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Human dermal papilla cells (DPCs), purchased from PromoCell (#C‐12071), were cultured in follicle DPC medium with supplement mix (PromoCell) and 0.1% anti‐antibiotics (Gibco). After informed consent was obtained, human outer root sheath cells (ORSs) were isolated (Yonsei University College of Medicine, 4‐2018‐0141). Human hair follicle was extracted, and the ORS part was cultured for 2‐3 days in a culture dish containing DPC medium with supplement mix. Next, proliferating cells were subcultured with ORS medium. The ORS was cultured in EpiLife medium (Gibco) with EpiLife™ Defined Growth Supplement (EDGS; Gibco) and 1% penicillin/streptomycin (Gibco). DPCs and ORSs for all experiments were used at passages 3‐4 and 1‐2, respectively. All cells were maintained at 37°C in a humidified 5% CO2 incubator.
8
Cultivation of Human Hair Follicle Cells
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Human outer root sheath (HORS) cells at passages of 3 to 5 were grown in an EpiLife medium consisting of 60 μM calcium (Gibco, New York, NY, USA), 1% EpiLife Defined Growth Supplement (EDGS) (Gibco), and 1% penicillin-Streptomycin (Gibco). Human dermal papilla (HDP) cells at passages of 4 to 8 were cultured with follicle dermal papilla cell growth medium consisting of a supplement mix (PromoCell, Heidelberg, Germany) and 0.1% antibiotic-antimycotic (Gibco). Cell incubation was conducted at 37 °C in 5% CO2.
9
Cell Culture Maintenance Protocol
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All cells listed in Table S3 were cultured at 37°C with 5% CO2 in the indicated growth medium with regular passage every 2 to 3 days. Stable cell lines were maintained in culture medium with an appropriate concentration of puromycin (10 μg/ml for CHO-K1 cells and pgsA-745 cells and 2 μg/ml for other cell types). Primary human epidermal keratinocytes were cultured on collagen-coated plates (catalog number R011K; Gibco) and maintained in EpiLife medium (catalog number M-EPI-500-CA; Gibco) supplemented with EpiLife defined growth supplement (EDGS) (catalog number S-012-5; Gibco).
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