Immortalized Human Gingival Keratinocytes (Gie-No3B11, abbreviated as iHGK) (Applied Biological Materials Inc., Richmond, BC, Canada) were grown on tissue culture flask for sensitive adherent cells (Sarstedt, Numbrecht, Germany) at 37 °C in an atmosphere of 5% CO2 using keratinocyte medium that consist in DMEM without magnesium and calcium (Gibco, Grand Island, NY, US)/Ham’s F12 (3/1) (Biowest), supplemented with 0.01 mg/mL of insulin (Sigma-Aldrich), 0.4 ng/mL of hydrocortisone (Sigma-Aldrich), 6.7 ng/mL of selenium (Sigma-Aldrich), 0.01 μg/mL of human epithelial growth factor (ThermoFisher Scientific), 1M HEPES-buffer (Biowest), 5.5 μg/mL of transferrin (Sigma-Aldrich), 10–10 M of cholera toxin (Sigma-Aldrich), 2 mM of L-glutamine (Sigma-Aldrich), 5% (v/v) of FBS embryonic stem cells tested (Biowest) and 100 µg/mL penicillin and 100 µg/mL streptomycin (Biowest). The culture medium was renewed twice per week. Immortalized Human Gingival Fibroblasts-hTERT (iHGF) were cultured as described in Section 2.3.1 . Both iHGK and iHGF cultures with 70–80% confluency were used for the construction of GTE as described in the next Section 2.4.2 .
Human epithelial growth factor
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human epithelial growth factor is a protein that stimulates the growth and proliferation of epithelial cells. It is a key regulator of various cellular processes, including cell migration, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Hydrocortisone, by Merck Group (2 mentions)
Human insulin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Selenium, by Merck Group (1 mentions)
Ham s f12 3 1, by Biowest (1 mentions)
Hepes buffer, by Biowest (1 mentions)
Insulin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Gie no3b11, by Applied Biological Materials (1 mentions)
Penicillin, by Biowest (1 mentions)
Streptomycin, by Biowest (1 mentions)
Bovine insulin, by Merck Group (1 mentions)
Tris edta buffer solution, by Merck Group (1 mentions)
Anhydrous mgso4, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium nutrient mixture f 12 dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
5 protocols using human epithelial growth factor
1
Culturing Immortalized Human Gingival Cells
2
Phytochemical Analysis and Comet Assay
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Milli-Q water was obtained from a Millipore unit. EtOH non denaturalized (99.9%) was bought from Dávila Villalobos S.L. (Valladolid, Spain), N2 (99.996%) from Linde Gas (Puçol, Spain), Fluorescein sodium salt from Vetec Química (Xerem Duque De Caxias, Rio de Janeiro, Brazil) and OxiSelect 96-Well Comet Assay Kit from bioNova scientific (Fremont, CA, USA). Methanol (MeOH, 99.9% LC-MS), n-hexane (95%), NaOH pellets, Dimethyl Sulfoxide (DMSO), and phosphoric acid were supplied by Panreac Quimica SLU (Barcelona, Spain), while commercial standards HT (≥98%), TY (≥99%) and OL (≥98%) by Extrasynthese (Genay, France). MgSO4 anhydrous, KI, NaBr, NaCl, Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), AAPH (2,2′-azobis(2-methylpropionamidine)dihydrochloride), gallic acid, catechin, Tris-EDTA buffer solution, and bovine insulin were purchased from Sigma-Aldrich (Madrid, Spain). Plastic culture flasks, plates, tips, and pipettes, Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) + GlutaMax, Dulbecco’s phosphate-buffered saline, fetal bovine serum, human epithelial growth factor, human insulin, penicillin, and streptomycin were supplied by Thermo Fisher Scientific (Rockford, IL, USA). CO2 (99.95%) was obtained by Carburos Metálicos (Barcelona, Spain).
3
Antibody-based protein analysis protocol
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Primary antibodies used in this study included: mouse anti-BrdU and mouse anti-β-catenin, BD Biosciences (San Jose, CA); rabbit anti-phospho-S6 ribosomal protein (Ser240/244) XP mAb, rabbit anti-phospho-mTOR (Ser2448) mAb, Cell Signaling (Danvers, MA); mouse anti- β-actin, Sigma-Aldrich (St. Louis, MO); rabbit anti-Sox9, Millipore (Bilerica, MA) and mouse anti-active β-catenin Milipore (Bilerica, MA). The secondary antibodies used were HRP-conjugated anti-rabbit IgG, Cell Signaling and HRP-conjugated anti-mouse IgG, Thermo Scientific (Cincinnati, OH). Dulbecco's Modifid Eagle Medium (DMEM), M199 and cosmic calf serum were from HyClone (Logan, UT). Tamoxifen, Alcian blue, human apo-transferin, hydrocortisone and sodium selenite were obtained from Sigma-Aldrich. Human epithelial growth factor was from Peprotech (Rocky Hill, NJ). Human insulin and 0.05% Trypsin-EDTA were from Invitrogen (Carlsbad, CA). Rapamycin (RAP) was purchased from LC Laboratories (Woburn, MA).
4
Organoid Culture of Lineage-traced AT1 Cells
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Lineage-traced AT1 cells were obtained following FACS and were then placed into an organoid growth medium containing DMEM F12 (Thermo Fisher Scientific) and growth factors including bovine pituitary extract, cholera toxin, FBS, gentamicin, retinoic acid, insulin, transferrin (all from Lonza), and human epithelial growth factor (Peprotech), as previously described (58 (link), 67 (link)). A total of 25,000 cells per well were plated onto 24-well plates that were coated with fibronectin (5 μg/cm2, Thermo Fisher Scientific) or Col4 (10 μg/cm2, Corning). To evaluate the effects of exogenous TGF-β ligand or inhibition of TGF-β signaling, TGF-β1 (7.5 ng/mL, Peprotech) or SB 431542 (10 μM, Abcam) were added at the time of plating. Cells were fixed in 2% PFA at days 2, 4, and 6 or collected in RNA lysis buffer. After fixation, cells were stained with anti-RAGE antibody (rat, R&D, MAB1179, 1:100) and the entire well was imaged with the EVOS FL Auto2 Imaging System at 20 × magnification. Only cells that were entirely within the image frame and were not adhered to an adjacent cell were assessed. Using Fiji software, cell area and roundness were quantified.
5
Exploring Human Renal Cell Dynamics
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The human renal tubular epithelial cell line HK-2 was obtained from ATCC (Manassas, VA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin (10,000 IU/mL and 10 mg/mL) were purchased from Gibco Inc. (Grand Island, NY). Fetal bovine serum (FBS) was obtained from Biological Industries (Beit Haemek, Israel). Human PTH (1–34) was acquired from Sigma Inc. (St. Louis, MO). Fasudil was provided by Tianjin Chase Sun Co. (Tianjin, China) and human epithelial growth factor was purchased from Peprotech Inc. (Rocky Hill, NJ). Anti-Rabbit IgG-FITC antibody, E-cadherin antibody and α-SMA antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Primers were designed and synthesized by Life Technologies (Invitrogen, Guangzhou, China). The reverse transcription reagent kit and the real-time polymerase chain reaction (PCR) reagents were purchased from TaKaRa Bio Inc. (Dalian, China).
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