Human gingival fibroblasts (HGFs) were obtained from Provitro AG (Berlin, Germany). The HGFs (passage 9) were cultured at 37 °C using Fibroblast Growth Medium (Provitro, Berlin, Germany) without antibiotics. Cells were grown to near confluency and irradiated in 175 cm2 bottles. Thereafter, cells were returned to the incubator or detached from the flasks by a brief treatment with trypsin/EDTA (PAA Laboratories, Dartmouth, MA, USA). After resuspension in a small volume of medium in a cell culture tube, these were snap-frozen in liquid N2 and stored at −86 °C until RNA preparation. With the exception of one combination of radiation dose and repair interval (0.5 Gy and 16 h), which was not used for quantitative analyses, experiments were performed in duplicates or triplicates (see Table Supplemental Digital Content 1, http://links.lww.com/HP/A52 which contains general information on the datasets).
Fibroblast growth medium
Manufactured by Provitro
Sourced in Germany
Fibroblast Growth Medium is a cell culture medium designed to support the growth and proliferation of fibroblast cells. It provides the necessary nutrients and growth factors to maintain fibroblast cultures in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by GE Healthcare (1 mentions)
Hepatocyte growth factor (hgf), by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Amphotericin, by Provitro (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Bis gma, by Evonik (1 mentions)
Quadriperm chambers, by Greiner (1 mentions)
4 protocols using fibroblast growth medium
1
Irradiated Human Gingival Fibroblasts
2
Culturing Primary Human Gingival Fibroblasts
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Primary human gingival fibroblasts (hGF) were purchased from Provitro GmbH, Berlin, Germany). Three different donors were used (range 19–47 years; male:female ratio 2:1). Provitro assures that cells were obtained ethically and legally and that all donors provided written informed consent. Cells were routinely cultured at 37 °C and 5% CO2, and maintained in fibroblast growth medium (Provitro GmbH) supplemented with foetal calf serum (10%; Provitro GmbH), amphotericin (50 ng/ml) and gentamicin (50 μg/ml). Experiments were performed with hGF between passages 7 and 8 after isolation and media was supplemented with ascorbic acid (100 μM; Sigma-Aldrich).
The different coins were placed in 96-well plates in sterile conditions. Three replicates for each donor were seeded at a density of 7.0 × 103 cells on each coin (n = 9). For cell adhesion experiments, four replicates from one randomly selected donor were used (n = 4).
In order to create inflammatory conditions, interleukin-1 beta (1 ng/ml) (IL-1β; R&D systems, Abingdon, UK) was added 1 d after cell seeding and kept until day 3, according to previous studies34 (link).
The different coins were placed in 96-well plates in sterile conditions. Three replicates for each donor were seeded at a density of 7.0 × 103 cells on each coin (n = 9). For cell adhesion experiments, four replicates from one randomly selected donor were used (n = 4).
In order to create inflammatory conditions, interleukin-1 beta (1 ng/ml) (IL-1β; R&D systems, Abingdon, UK) was added 1 d after cell seeding and kept until day 3, according to previous studies34 (link).
3
Genotoxicity of Dental Monomers
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The composite components Bis-GMA (CAS-No. 1565-94-2), TEGDMA (CAS-No. 109-16-0) and HEMA (CAS-No. 868-77-9) were obtained from Evonik Röhm (Essen, Germany), GMA (CAS-No. 106-91-2) and antioxidant NAC were obtained from Sigma-Aldrich (Steinheim, Germany). All studied substances are listed in Table 1. In addition to the monomers we used acrylamide (ACR) as a marker substance for epoxide formation, because it is known that metabolization of ACR can form an epoxide compound, glycidamide, which is highly reactive 47) . ACR (CAS No. 79-06-1) was obtained from Sigma-Aldrich (Table 1).
Bis-GMA, GMA, HEMA, TEGDMA and ACR were deployed in a dilution series of the pure substance with Fibroblast Growth Medium (Provitro, Berlin, Germany)+dimethyl sulfoxide (DMSO, 99% purity; Merck, Darmstadt, Germany) at a final concentration of 0.5%.
Controls contained: 1) medium+0.5%DMSO, 2) medium+0.5%DMSO+10 mM NAC. The antioxidant concentration used was chosen on the basis that previous studies revealed a reduction of the TEGDMA and HEMA genotoxicity at a dose of 10 mM NAC 11, 48, 49) . As in our previous experiments we used cells exposed to 1 mM hydrogenperoxide (Sigma-Aldrich) dissolved in medium 28) and 0.5 Gy X-ray exposed cells 30) as a positive controls (not shown; see refs. 28, 30).
Bis-GMA, GMA, HEMA, TEGDMA and ACR were deployed in a dilution series of the pure substance with Fibroblast Growth Medium (Provitro, Berlin, Germany)+dimethyl sulfoxide (DMSO, 99% purity; Merck, Darmstadt, Germany) at a final concentration of 0.5%.
Controls contained: 1) medium+0.5%DMSO, 2) medium+0.5%DMSO+10 mM NAC. The antioxidant concentration used was chosen on the basis that previous studies revealed a reduction of the TEGDMA and HEMA genotoxicity at a dose of 10 mM NAC 11, 48, 49) . As in our previous experiments we used cells exposed to 1 mM hydrogenperoxide (Sigma-Aldrich) dissolved in medium 28) and 0.5 Gy X-ray exposed cells 30) as a positive controls (not shown; see refs. 28, 30).
4
Culturing and Exposing Human Gingival Fibroblasts
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The HGFs (Cat-No.: 1210412) were obtained from Provitro, Cell-Lining (Berlin, Germany). The HGFs (passage 9) were cultured in 175 cm 2 cell culture flasks using Fibroblast Growth Medium (Provitro) in an incubator with 5% CO 2 at 100% humidity and 37°C. HGFs were seeded directly on glass slides (Super Frost ® Plus, Menzel Gläser, Braunschweig, Germany) in quadriPERM ® chambers (GreinerBio-One, Frickenhausen, Germany). The method of cell culturing was as described previously 30) . After HGFs reached 70% confluence, cells were exposed for 6 h with the tested substances alone or together with the antioxidant (Table 1).
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