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.
Rt4 d6p2t
Manufactured by American Type Culture Collection
Sourced in United States
The RT4-D6P2T is a laboratory equipment used for cell culture applications. It serves as an incubator, maintaining a controlled environment for the growth and maintenance of cell lines. The core function of this product is to provide a temperature- and humidity-regulated environment to support the optimal conditions for cell culture processes.
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Lab products found in correlation
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Lonza (2 mentions)
Streptomycin, by Lonza (2 mentions)
Hdf a, by ScienCell (1 mentions)
Dmem f12, by Harvard Bioscience (1 mentions)
Dmem f12, by R&D Systems (1 mentions)
N 2 max media, by R&D Systems (1 mentions)
Recombinant human egf, by R&D Systems (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
H peo, by Colorcon (1 mentions)
L peo, by Merck Group (1 mentions)
Trypsin edta solution, by Avantor (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
8 protocols using rt4 d6p2t
1
Culturing Primary Human Dermal Fibroblasts, Rat Schwannoma, and Human Astrocytes
2
Electrospinning of Polymeric Solutions for Neuronal Cell Culture
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Deacetylated gellan gum (GG, Gelrite®; Kelco Division of Merck & Co., Rahway, New Jersey, USA), spermidine trihydrochloride (SP; Sigma Aldrich, Milan, Italy), gelatin from porcine skin (GL, gel strength 90–110 g Bloom, Type A; Sigma Aldrich, Milan, Italy), poly(ethylene oxide) of high molecular weight (h-PEO, MW = 4000 kDa; Colorcon, Dartford, United Kingdom), poly(ethylene oxide) of low molecular weight (l-PEO, MW = 600 kDa; Sigma Aldrich, Milan, Italy) and Kolliphor P407 poloxamer (P407; Sigma Aldrich, Milan, Italy) were used for the preparation of the polymeric solutions to be electrospun.
For the experiments with Neuronal Schwann cells RT4-D6P2T (ATCC CRL-2768), the materials hereafter reported were used. Dulbecco’s Modified Eagle’s Medium (ATCC-30-2002) was purchased from LGC Standards S.r.L. (Milan, Italy), while inactivated Fetal Bovine Serum (FBS) from Biowest (Nuaillé, France). Phosphate Buffer Solution (PBS), antibiotic/antimycotic solution (100X; stabilized with 10,000 units penicillin, 10 mg streptomycin, and 25 g amphotericin B per mL) and trypsin–EDTA solution were purchased from VWR (Radnor, Pennsylvania, USA). Dimethyl sulfoxide (DMSO), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Trypan Blue solution were purchased from Sigma-Aldrich (Milan, Italy).
For the experiments with Neuronal Schwann cells RT4-D6P2T (ATCC CRL-2768), the materials hereafter reported were used. Dulbecco’s Modified Eagle’s Medium (ATCC-30-2002) was purchased from LGC Standards S.r.L. (Milan, Italy), while inactivated Fetal Bovine Serum (FBS) from Biowest (Nuaillé, France). Phosphate Buffer Solution (PBS), antibiotic/antimycotic solution (100X; stabilized with 10,000 units penicillin, 10 mg streptomycin, and 25 g amphotericin B per mL) and trypsin–EDTA solution were purchased from VWR (Radnor, Pennsylvania, USA). Dimethyl sulfoxide (DMSO), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Trypan Blue solution were purchased from Sigma-Aldrich (Milan, Italy).
3
Rat Schwann Cell-like Culture Protocol
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The present study was performed using the rat Schwann cell-like culture RT4-D6P2T (ATCC number CRL-2768) obtained from the American Type Culture Collection (Rockville, MD, USA).
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% of heat-inactivated fetal bovine serum (FBS), 100U/ml penicillin, and 100 μg/ml streptomycin (Lonza, Italy). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Cells were grown to reach about 80–85% confluence in media containing 10% FBS and subjected to different treatments as described in the related subsections.
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% of heat-inactivated fetal bovine serum (FBS), 100U/ml penicillin, and 100 μg/ml streptomycin (Lonza, Italy). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Cells were grown to reach about 80–85% confluence in media containing 10% FBS and subjected to different treatments as described in the related subsections.
4
Rat Schwannoma Cell Culture Protocol
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Cell culture reagents were purchased from Gibco (Life Technologies, Carlsbad, CA) unless otherwise noted. Rat Schwannoma cells, RT4-D6P2T (ATCC, Manassas, VA), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (P-S). Cells were incubated at 37°C in an atmosphere of 5% CO2. Cell experiments were performed using passage 5–10 cells.
5
Cell Culture for Proliferation and ICC
6
Evaluating Schwann Cell Viability on AuNRs
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An immortalized Schwann cell line, RT4-D6P2T was obtained from the American Type Culture Collection. A second passage of RT4-D6P2T cells, were incubated for 7 days over the AuNRs substrates, and used in the experiments described below. Tissue culture treated Thermanox cover slips were used as positive controls. Cells were incubated at 37 °C (5% CO2, 95% air) in DMEM medium supplemented with 10% fetal bovine serum (ATCC, 63310972) and 1% penicillin/ streptomycin. The medium was changed every two days.
RT4-D6P2T cells seeded on the AuNRs were evaluated for their viability, adherence, morphology and the expression of Schwann cell protein markers using the WST-1 and immunofluorescence assays, respectively.
RT4-D6P2T cells seeded on the AuNRs were evaluated for their viability, adherence, morphology and the expression of Schwann cell protein markers using the WST-1 and immunofluorescence assays, respectively.
7
Culturing Rat Spiral Ganglion Neurons
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Rat SCs line RT4-D6P2T was purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose (Life Technologies, Carlsbad), supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and were incubated at 37°C in a humidified atmosphere containing 5% CO2.16 (link)
A total of 12 Sprague-Dawley rats (postnatal day 3–5) were provided by the Animal Center of the Academy of Military Science of the Chinese PLA. Cochleae were isolated after rats were decapitated, and SGNs were dissociated as previous described.17 (link) The SGNs were cultivated in DMEM high glucose (Life Technologies), 25 mM HEPES (Life Technologies), 30 U/mL penicillin (Grünenthal GmbH, Aachen), 3 μL/mL N2 supplement (Life Technologies) and 5 μg/mL insulin (Sigma-Aldrich, St. Louis).18 (link) SGNs were incubated in a humidified atmosphere with 5% CO2 at 37°C. The mere killing of rats for tissue analyses was approved by our local ethics committee. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Precautions were taken to minimize suffering and the number of animals used in each experiment.
A total of 12 Sprague-Dawley rats (postnatal day 3–5) were provided by the Animal Center of the Academy of Military Science of the Chinese PLA. Cochleae were isolated after rats were decapitated, and SGNs were dissociated as previous described.17 (link) The SGNs were cultivated in DMEM high glucose (Life Technologies), 25 mM HEPES (Life Technologies), 30 U/mL penicillin (Grünenthal GmbH, Aachen), 3 μL/mL N2 supplement (Life Technologies) and 5 μg/mL insulin (Sigma-Aldrich, St. Louis).18 (link) SGNs were incubated in a humidified atmosphere with 5% CO2 at 37°C. The mere killing of rats for tissue analyses was approved by our local ethics committee. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Precautions were taken to minimize suffering and the number of animals used in each experiment.
8
Rat Schwann Cell Culture Protocol
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The present study was performed using the rat Schwann cell line RT4-D6P2T (ATCC number CRL-2768) obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% of heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Lonza, Milan, Italy). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells were grown to reach about 80–85% confluence in media containing 10% fetal bovine serum (FBS) before treatment with lipopolysaccharides (LPS) from Escherichia coli O111:B4 (cat no# L4391, Sigma Aldrich, St. Louis, MO, USA) at the concentrations and time regimens described in the related subsections. LPS was diluted in water from the stock solution and stored at −80 °C until needed.
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