Human keratinocytes cells (ATCC PCS-200-011) were grown in Dermal Cell Basal Medium supplemented with Keratinocyte Growth Kit (ATCC); whilst human dermal fibroblast cells were kindly supplied by Prof. Stephens40 (link) from Cardiff University and grown in Minimum Essential Medium Eagle (MEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS). Both media were supplemented with 1% penicillin–streptomycin (PS). Both cells lines were incubated at 37 °C in a humidified air atmosphere with 5% CO2; media were changed twice per week. 96 well plates were seed with 150 μL containing 10,000 viable cells as determined through trypan blue staining and incubated for 5 days at 37 °C in a humidified air atmosphere with 5% CO2; media were changed after 2 days.
Keratinocyte growth kit
Manufactured by American Type Culture Collection
Sourced in United States
The Keratinocyte Growth Kit is a laboratory product that provides a defined, serum-free medium formulation to support the growth and proliferation of human epidermal keratinocytes in cell culture. The kit contains a basal medium and a supplement solution that must be combined to create the complete growth medium.
Automatically generated - may contain errors
Lab products found in correlation
Dermal cell basal medium, by American Type Culture Collection (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Melanocyte growth kit, by American Type Culture Collection (4 mentions)
Pcs 200 014, by American Type Culture Collection (3 mentions)
Pcs 200 030, by American Type Culture Collection (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin streptomycin, by Welgene (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Pcs 200 040, by American Type Culture Collection (2 mentions)
Ccd 986sk, by American Type Culture Collection (2 mentions)
Iscove s modified dulbecco s medium, by Welgene (2 mentions)
Penicillin streptomycin amphotericin b solution, by American Type Culture Collection (2 mentions)
Thp 1, by American Type Culture Collection (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Pcs 200 011, by American Type Culture Collection (2 mentions)
39 protocols using keratinocyte growth kit
1
Culturing Human Skin Cells for Experiments
2
Culturing HGECs and BMMCs for P. gingivalis stimulation
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HGECs were cultured according to the manufacturer’s protocol (PCS-200-014, American Type Culture Collection (ATCC), Manassas, VA, USA). The cells were sub-cultured in Dermal Cell Basal Medium (PCS-200-030, ATCC, Manassas, VA, USA) supplemented with Keratinocyte Growth Kit (PCS-200-040, ATCC, Manassas, VA, USA) and 100 U penicillin/streptomycin (P4333-100ML, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in humidified air with 5% CO2. A confluent culture of HGECs was stimulated with P. gingivalis (10% formalin-fixed, strain 3327, ATCC, Manassas, VA, USA) (107 colony-forming units/mL) for 12 h to examine mRNA expression. As a negative control, HGECs were cultured without bacterial stimulation. P. gingivalis treatment was performed as previously described [20 (link)].
Bone marrow mononuclear cells (BMMCs) were collected from the femurs and tibias of C57BL/6J Jcl mice by density gradient centrifugation with Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) in complete alpha-modified Eagle’s minimum essential medium (Sigma-Aldrich), containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (penicillin, streptomycin, and gentamicin; Invitrogen, Carlsbad, CA, USA).
Bone marrow mononuclear cells (BMMCs) were collected from the femurs and tibias of C57BL/6J Jcl mice by density gradient centrifugation with Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) in complete alpha-modified Eagle’s minimum essential medium (Sigma-Aldrich), containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (penicillin, streptomycin, and gentamicin; Invitrogen, Carlsbad, CA, USA).
3
Cell Culture Conditions for Keratinocytes and Carcinoma Cells
4
Culturing Epidermoid Carcinoma and Keratinocytes
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Epidermoid carcinoma cells A431 were cultivated as previously described (Del Favero et al. 2018a (link)) in Minimum Essential Medium (MEM) with l -glutamine (4.5 g/L), 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin and maintained in controlled humidified incubators at 37 °C and 5% CO2. Primary Epidermal Keratinocytes (HEKn; Normal, Human, Neonatal Foreskin ATCC® PCS-200-010™) were cultivated according to the specification of the supplier in Dermal Cell Basal Medium (ATCC® PCS-200-030™) including Keratinocyte Growth Kit (ATCC® PCS-200-040™). If not otherwise specified, cell culture reagents were purchased from GIBCO Invitrogen (Karlsruhe, Germany), Sigma-Aldrich Chemie GmbH (Munich, Germany), Sarstedt AG & Co (Nuembrecht, Germany), VWR International GmbH (Vienna, Austria) and Thermo Fisher Scientific GmbH (Vienna, Austria). Commercially available DON was purchased from Romer Labs (Tulln, Austria). Solid substance was dissolved in dimethyl sulfoxide (DMSO; Carl Roth GmbH, Karlsruhe, Germany) and diluted in cell culture media (1:1000). Respective DMSO concentration (0.1%) was used as negative/solvent control (controls, CONT).
5
Cell Culture Protocols for Cancer and Normal Cell Lines
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MDA-MB-231 and BT-20 cell lines were obtained from late Dr. Gary Kruh (University of Chicago, Illinois). Human primary epithelial gingival keratinocytes (HPK), and normal colon fibroblast CRL1459 cells were purchased from American Type Culture Collection (ATCC#PCS-200-014, and ATCC#CCD-18Co—CRL-1459, respectively, Manassas, VA, USA). These cells were grown as an adherent monolayer with Dulbecco’s Modified Eagle’s Medium (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Biofluids Technologies, Port Richey, FL, USA), 2 mM of L-glutamine and 100 U/mL of penicillin and 100 µg/mL of streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described [42 (link)]. HPKs cells were cultured in dermal cell basal medium (ATCC# PCS-200-030) supplemented with factors from Keratinocyte growth kit (ATCC# PCS-200-040) as per manufacture’s protocol. These cells were cultured in a humidified incubator containing 5% CO2 at 37 °C. All cells were assessed and confirmed to be free of fungi and mycoplasma. Cells were obtained from frozen stocks and cell passaging (up to P4) was performed at 80% cell confluency trypsin + 2.2 mM EDTA in phosphate-buffered saline (PBS).
6
Establishment of Oral Cancer Cell Lines
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Human OSCC cell lines derived from oral cancer (SAS, HSC-2, HSC-3, Ca9-22, OSC-19, OSC-20, SAT, and KON) were purchased from the National Institute of Biomedical Innovation (Osaka, Japan). The HOC-31354 (link) and TSU55 (link) cell lines were kindly provided by Professor Kawashiri (Kanazawa University). HNOK cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA; PCS-200-014). SAS-R and HSC-2-R, which were established from SAS and HSC-2 cells, were used as the CRR cell lines. The CRR cell lines were produced by exposing cells to gradually increasing X-ray doses.5 (link) The OSCC cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; D6429; Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37°C and 5% CO2. HNOK cells were cultured in dermal cell basal medium (PCS-200-030; ATCC) supplemented with the Keratinocyte Growth Kit (PCS-200-040; ATCC). CRR cells continued to proliferate under a daily IR dose of 2 Gy for >30 days in vitro.
7
Culturing Primary Human Skin Cells
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Primary human epidermal keratinocytes (HEKn; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in growth medium (GM) consisting of dermal cell basal medium (ATCC) supplemented with a keratinocyte growth kit (ATCC). Cultures were maintained at 37 °C and 5% CO2.
Human dermal fibroblasts (CCD-986Sk; ATCC) were cultured in medium composed of Iscove’s Modified Dulbecco′s Medium (Welgene, Gyeongsan, Korea), fetal bovine serum (GibcoTM, Thermo Fisher Scientific, Rockford, IL, USA), and 1% penicillin/streptomycin (Welgene). Cells were also maintained at 37 °C and 5% CO2.
Human dermal fibroblasts (CCD-986Sk; ATCC) were cultured in medium composed of Iscove’s Modified Dulbecco′s Medium (Welgene, Gyeongsan, Korea), fetal bovine serum (GibcoTM, Thermo Fisher Scientific, Rockford, IL, USA), and 1% penicillin/streptomycin (Welgene). Cells were also maintained at 37 °C and 5% CO2.
8
Cell Culture Protocols for Cancer and Endothelial Cells
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L3.6pl tumor cells were obtained from Dr Craig Logsdon (University of Texas, MD Anderson Cancer Center, Houston, TX, USA), grown in minimal essential medium (MEM) with 10% fetal bovine serum and penicillin/streptomycin, and passaged every 72 h. All experiments involving L3.6pl cells were completed from passage 10–20. HUVEC were purchased from Lonza (Basel, Switzerland), grown in endothelial growth media supplemented with an endothelial cell SingleQuot kit (Lonza), and characterized as >90% double-positive for CD31/CD105 at passage four. HUVEC were passaged every 72 h and used between passages two through eight. Human primary epidermal keratinocytes (American Type Culture Collection (ATCC), Manassas, VA, USA) and COCA mouse epidermal keratinocytes (Sigma Aldrich, St. Louis, MO, USA) were cultured in dermal cell basal media supplemented with a keratinocyte growth kit (ATCC). All cell lines were tested for mycoplasma contamination and maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
9
Cell Culture Conditions for Various Cancer and Normal Cell Lines
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CAPAN2, PANC 10.05, PANC 03.27, HPAC, IMR-90, BJ and HEKa (primary epidermal keratinocytes, adult) cell lines were purchased from ATCC. RPE-1 cells were purchased from Clontech. hTERT-HPNE (HPNE) cells were provided by Dr. Steve Elledge. MCF10A cells were provided by Dr. Jayanta Debnath. HPD cells27 (link) were provided by Dr. Abigail Miller. PANC 10.05 and PANC 03.27 cells were grown in RPMI supplemented with 10% FBS, Penicillin-Streptomycin and L-glutamine. CAPAN2 cells were grown in McCoy’s 5A supplemented with 10% FBS, Penicillin-Streptomycin and L-glutamine. HPAC, HPD and RPE-1 cells were grown in DMEM supplemented with 10% FBS, Penicillin-Streptomycin and L-glutamine. IMR-90 and BJ cells were grown in EMEM supplemented with 10% FBS, Penicillin-Streptomycin and L-glutamine. HPNE cells were grown in 75% DMEM and 25% M3 Base Medium (Incell Corp., #M300F) supplemented with 10% FBS, Penicillin-Streptomycin and L-glutamine. MCF10A cells were grown in DMEM/F12 supplemented with 5% horse serum, Penicillin-Streptomycin, 20 ng/ml EGF, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin and 10 μg/ml insulin. HEKa cells were grown in Dermal Cell Basal Medium (ATCC, #50-189-650FP) supplemented with a keratinocyte growth kit (ATCC, #50-189-651FP). All cell lines were grown at 37°C and 5% CO2.
10
Cell Culture Protocols for Cancer and Epithelial Cell Lines
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U251SP cells (human glioblastoma cell line) and MCF10A cells (human mammary epithelial cell line) were grown in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)-streptomycin (100 μg/mL; P/S). SiHa cells (a human cervical cancer cell line) and SK-OV-3 cells (a human ovarian cancer cell line) were grown in Roswell Park Memorial Institute 1640 (Sigma-Aldrich) supplemented with 10% FBS and P/S). Human neonatal keratinocyte cells (purchased from ATCC, Manassas, VA) were grown using a keratinocyte growth kit (purchased from ATCC, Manassas, VA) under an atmosphere of 5% CO2 at 37 °C.
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