Parental TAP deficient T2 cells (ATCC, Manassas, Virginia) as well as recombinant HLA‐A*24:02 transduced T2 cells were cultured in RPMI 1640 (Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, heat inactivated) and 2 mM L‐glutamine (c‐c Pro, Oberdorla, Germany). HEK293T‐cells were cultured for transfection in DMEM (Life Technologies, Darmstadt, Germany) supplemented with 10% FBS (heat inactivated), 2 mM L‐glutamine and 1 mg/mL Geneticin (Life Technologies, Darmstadt, Germany). NHDF‐c cells were cultured in DMEM supplemented with 10% FBS (heat inactivated) and 2 mM L‐glutamine. Parental fibroblast BJ cells (ATCC, Manassas, Virginia) as well as recombinant HLA‐A*24:02 transduced BJ cells were cultured in DMEM supplemented with 20% medium 199, 10% FBS (heat inactivated) and 2 mM L‐glutamine. All HCMV infections were performed with strain AD169 (ATCC). For the preparation of virus stock, NHDF‐c ells (PromoCell, Heidelberg, Germany) were infected and cultured until 100% cytopathic effect was seen. The virus was pelleted and stored at −80°C. To determine the virus titer, plaque‐assay was performed on NHDF‐c cells cultured in carboxymethyl cellulose (CMC) medium.
Nhdf cells
Manufactured by PromoCell
Sourced in Germany, Australia
NHDF cells are primary human dermal fibroblasts derived from normal human skin tissue. These cells are designed for use in cell-based assays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Amaxa human dermal fibroblasts nucleofector kit, by Lonza (1 mentions)
Celltiter glo luminescent, by Promega (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions)
Nci n87, by American Type Culture Collection (1 mentions)
Genomic dna extraction kit, by Corning (1 mentions)
Trypsin versene edta mixture, by Lonza (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Dmem f12 medium, by Lonza (1 mentions)
Penicillin streptomycin amphotericin b mixture, by Lonza (1 mentions)
6 protocols using nhdf cells
1
Recombinant HLA-A*24:02 T cell and Fibroblast Cultures
2
Modulation of NHDF Cell Inflammation
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For the treatment, the NHDF cells (PromoCell, Germany) were grown in six-well plates for 24 h. After fresh medium had been added, the inflammatory state was induced with LPS (100 ng/ml final concentration in the culture) in PBS. The incubation lasted 6 h, before the dressing extract and control samples were added and incubated for 24 h. An ethanol negative control and CBD and β-sitosterol standard positive controls were also included pure CBD in final concentrations corresponding to flax preparations (0.1 µg/ml), and β-sitosterol 1.82 µg/ml. The ethanol concentration was 0.1%. The cells were harvested after 24 h of treatment and directly used for RNA isolation.
To determine the activity of the histone acetyltransferase/histone deacetylase genes, NHDF cells were grown in a cell culture dish (100 mm) for 24 h. After adding fresh medium, the incubation was continued for 6 h, and after the 3-hydroxybutyrate monomer at the final concentration of 50, 75 and 200 µM in PBS was added. Afterwards, the flax fabric (containing 50 µmol of PHB) was added and incubation was continued for 24 h at 37°C. The cells were harvested after 24 h of treatment and directly used for total RNA isolation.
To determine the activity of the histone acetyltransferase/histone deacetylase genes, NHDF cells were grown in a cell culture dish (100 mm) for 24 h. After adding fresh medium, the incubation was continued for 6 h, and after the 3-hydroxybutyrate monomer at the final concentration of 50, 75 and 200 µM in PBS was added. Afterwards, the flax fabric (containing 50 µmol of PHB) was added and incubation was continued for 24 h at 37°C. The cells were harvested after 24 h of treatment and directly used for total RNA isolation.
3
Overexpression of SHOX in Dermal Fibroblasts
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Normal human dermal fibroblasts (NHDF cells; PromoCell: C12300) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 5% CO2-humidified incubator at 37°C. 1 × 106 NHDF cells were transfected with 3.5 μg pcDNA4/TO-SHOX-WT using the Amaxa® Human Dermal Fibroblasts Nucleofector® Kit (Lonza) according to the manufacturer’s instructions. In control experiments, NHDF were transfected with 3.5 μg pcDNA4/TO-SHOX-HM, which encodes a homeodomain-mutant of SHOX leading to the amino acid exchange Y141D.
4
Melanoma Cell Lines and Cytotoxicity Assays
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RPMI-1640 media, heat-inactivated foetal bovine serum (FBS), streptomycin, and penicillin, were obtained from Thermo Fisher Scientific (Melbourne, Australia). 3-(4,5-Di methylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), annexin V-FITC (Thermofisher, 11-8005-74) and PI suspended in annexin-binding buffer (Thermofisher, 00-6990-42) were purchased from Thermofisher (Melbourne, Australia). The propidium iodide flow cytometry kit (Abcam139418) was purchased from Abcam (Melbourne, Australia), while the cellTiter-Glo® Luminescent was purchased from Promega (Melbourne, Australia).
Human melanoma MM418-C1 (primary (1°) melanoma possessing the oncogenic BRAFV600E mutation), MM329 (1° melanoma possessing wild type BRAF (BRAFWT)) and MM96L (metastatic or secondary (2°) melanoma possessing the oncogenic BRAFV600E mutation) cells, human epithermal melanocytes (HEM), human immortalized keratinocytes (HaCaT), and neonatal human dermal fibroblasts (NHDF) were used in this study. MM418-C1, MM329, MM96L, and HaCaT cells were kindly supplied by Prof Nichola Hayward and Peter Parsons, Queensland Institute of Medical Research, Brisbane, Australia. NHDF cells were purchased from Promocell (Banksia Scientific, Brisbane, Australia).
Human melanoma MM418-C1 (primary (1°) melanoma possessing the oncogenic BRAFV600E mutation), MM329 (1° melanoma possessing wild type BRAF (BRAFWT)) and MM96L (metastatic or secondary (2°) melanoma possessing the oncogenic BRAFV600E mutation) cells, human epithermal melanocytes (HEM), human immortalized keratinocytes (HaCaT), and neonatal human dermal fibroblasts (NHDF) were used in this study. MM418-C1, MM329, MM96L, and HaCaT cells were kindly supplied by Prof Nichola Hayward and Peter Parsons, Queensland Institute of Medical Research, Brisbane, Australia. NHDF cells were purchased from Promocell (Banksia Scientific, Brisbane, Australia).
5
Cell Culture and Authentication of Gastric Cancer Cell Lines
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The NCI-N87 cells were cultured and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at a temperature of 37 °C. The cells were passaged 2−3 times per week when they reached 90-100% confluency. The human gastric cancer cell line NCI-N87 was obtained from ATCC®, CRL-5822 ™. The normal human dermal fibroblasts NHDF cells were purchased from PromoCell® and cultured in DMEM medium with 10% FBS. The human gastric cancer SNU-216 cells were purchased from BOHUI Biotechnology Co., Ltd, and were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The N87 and NFDH cell lines used were directly used without any authentication. The SNU-216 cells were authenticated through Short tandem repeat (STR) testing, performed by Shanghai biowing applied biotechnology Co., Ltd. In detail, DNA extraction will be performed using the Axygen Genomic DNA Extraction Kit. Subsequently, DNA samples will undergo amplification using the 21-STR amplification scheme, followed by the detection of the STR loci and the gender gene Amelogenin on the ABI3730XL genetic analyzer. Appropriate positive and negative controls were run and confirmed for the sample. All the cell lines tested negative for mycoplasma contamination.
6
Cytotoxicity Assay with NHDF Cells
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For cytotoxicity tests, normal human dermal fibroblasts (NHDF) cells (from PromoCell) at a passage number lower than 10 were grown in T25 culture flasks with 10 mL of DMEM:F12 medium (from Lonza) supplemented with 10% fetal bovine serum (FBS, Gibco), 1 mM sodium pyruvate (Lonza) and 1 % Penicillin-Streptomycin-Amphotericin B mixture (10K/10K/25 µg in 100 mL, Lonza). Cell growing was done at 37 °C and 5 % CO 2 under humidified atmosphere and medium was changed with fresh one every 4 days. NHDF cells were harvested by trypsinization with Trypsin-Versene (EDTA) mixture (Lonza), washed with phosphate buffered saline (PBS, Invitrogen) centrifuged at 200 rpm for 5 min, counted and seeded at a density of 5x10 3 cells/well in 96 well plates with 100 µL/well of the above specified medium. Cells were incubated and were allowed to adhere in humidified atmosphere at 37°C until the next day.
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