Primary human adult dermal fibroblasts (HDF-a, catalog number 2320, ScienCell, Carlsbad, CA, USA) were cultured in DMEM/F12 (Biochrom, Berlin, Germany) containing 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), GlutaMAX (2mM L-alanyl-L-glutamine, Life Technologies) and 1% antibiotic antimycotic solution Sigma-Aldrich, Saint-Louis, MO, USA). The rat schwannoma cell-line (RT4-D6P2T, catalog number CRL-2768, ATCC, Manassas, VA, USA) was cultured in high-glucose DMEM (Life Technologies) with 10% FBS. Human astrocytes (HA, catalog number 1800, ScienCell) were cultured in DMEM/F12 containing 10% FBS to which were added 1% N-2 MAX Media Supplement R&D Systems, Minneapolis, MN, USA) and 20 ng/mL recombinant human EGF (R&D Systems). All cell lines were cultured in a humidified incubator with 5% CO2 at 37°C and medium was changed every other day. When cells reached 80% confluency, the cells were passaged using 0.05% trypsin/EDTA in PBS (Life Technologies). At passage 5, the cells were fixed in 1% formaldehyde in PBS (15 minutes), washed with PBS and processed for immunohistochemistry or lysed in RNA-Bee and stored at -20°C for RNA isolation.
Hdf a
Manufactured by ScienCell
Sourced in United States
The HDF-a is a specialized laboratory equipment used for the isolation and purification of human dermal fibroblasts (HDFs) from skin tissue samples. It is a core component in cell culture research and applications that require the use of primary HDFs.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Harvard Bioscience (1 mentions)
Rt4 d6p2t, by American Type Culture Collection (1 mentions)
Dmem f12, by R&D Systems (1 mentions)
N 2 max media, by R&D Systems (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Recombinant human egf, by R&D Systems (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Platinum quantitative pcr supermix udg with rox, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Picopure rna isolation kit, by Arcturus (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum fcs, by Greiner (1 mentions)
Saos 2, by American Type Culture Collection (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Collagenase ia, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
5 protocols using hdf a
1
Culturing Primary Human Dermal Fibroblasts, Rat Schwannoma, and Human Astrocytes
2
Quantitative RT-PCR for Gene Expression
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Total RNA was isolated with RNeasy Plus mini kit® (Qiagen, Maryland, USA) or PicoPure™ RNA Isolation Kit (Arcturus Bioscience, CA, USA) according to the manufacturer's instructions. An aliquot of total RNA was reverse-transcribed by using an oligo (dT) primer. A cDNA template was amplified using the Platinum Quantitative PCR SuperMix-UDG with ROX (Invitrogen) and ABI7900HT Sequence Detection System (Applied Biosystems). Fluorescence was monitored during every PCR cycle at the annealing step. The authenticity and size of the PCR products were confirmed using a melting curve analysis (using software provided by Applied Biosystems) and a gel analysis. mRNA levels were normalized using β-actin as a housekeeping gene. The expression level in the photoreceptor-directed fibroblasts, HDF-a (Human Dermal Fibroblasts-adult, ScienCell Research Laboratories), 2 weeks post-transduction was used as a reference. The design of the PCR primer sets is shown in our previous paper [23 (link)] and Table S2 (Additional file 2 ).
3
Isolation and Culture of Primary Cells
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Collected tissue samples were minced and incubated at 37°C for 2 h in α‐MEM (Gibco, Carlsbad, CA) with collagenase I A (2 mg/ml; Sigma–Aldrich, St Louis, MO). The cell suspension was then centrifuged and washed twice in α‐MEM supplemented with 10% Fetal Calf Serum (FCS; Greiner Bio One, Kremsmünster, Austria). Cells were cultured in petri‐dishes in α‐MEM supplemented with 10% FCS, 1% Glutamax (Gibco), 3% penicillin and streptomycin (Gibco), and 25 μg/ml Amphotericin B (Gibco) for 72 h. Thereafter, cells were cultured in the same medium but without Amphotericin B. When cultures reached 90% confluence, the cells were transferred to 75 cm2 flasks. For the experiments cells from passage 1 or 2 were used.
The human osteosarcoma cell line SaOS‐2 (ATCC, Manassas, VA) and the human dermal fibroblast cell line (HDF‐a; ScienCell, Carlsbad, CA) were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin and streptomycin (Gibco). These cell lines were used as positive and negative controls in the experiments, respectively.
The human osteosarcoma cell line SaOS‐2 (ATCC, Manassas, VA) and the human dermal fibroblast cell line (HDF‐a; ScienCell, Carlsbad, CA) were cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin and streptomycin (Gibco). These cell lines were used as positive and negative controls in the experiments, respectively.
4
Fibroblast response to glucose
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The adult human dermal fibroblasts (HDF-a, ScienCell, USA) were obtained from ScienCell Research Laboratories. The cells cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) were supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Gibco, UK). All cultures were maintained in a humidified incubator at 37°C and 5% CO2 atmosphere. The 70% confluent fibroblast cultures were maintained 24 hours in the growth medium for synchronization of the cell cycle. For further experiments, cells were treated with 3 different concentrations of glucose: 5.5 mM, 25 mM, and 60 mM for 72 hours.
5
Dermal Fibroblast and Iris Cell Cultures
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Three strains of cultured human dermal fibroblasts were used: one was obtained from Lonza (NHDF), another was from Promo Cell (NHDF) and the other was from ScienCell (HDF-a). These three kinds of fibroblasts were designated as Fib#1, Fib#2 and Fib#3, respectively. The cells were cultured in the recommended medium by the manufactures (FGM-2 Bullet kit, Fibroblast Growth Medium Kit, and Fibroblast Medium, respectively). Iris cells were obtained as previously reported (Seko et al. 2012 (link)) with the approval (approval number, #156) of the Ethics Committee of the National Institute for Child and Health Development (NCCHD), Tokyo. The ethics committees of the NCCHD and National Rehabilitation Center for Persons with Disabilities specifically approved this study. Signed informed consent was obtained from donors, and the surgical specimens were irreversibly de-identified. All experiments handling human cells and tissues were carried out in line with the Tenets of the Declaration of Helsinki. The iris cells were cultured in the growth medium [Dulbecco's modified Eagle's medium (DMEM)/Nutrient mixture F12 (1:1) supplemented with 10% fetal bovine serum, insulin–transferrin–selenium, and MEM-NEAA (GIBCO)].
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