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2 protocols using amphotericin

1

Culturing Diverse Cell Lines for Experiments

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HEK293T, MCF7, and C2C12 cells were grown in DMEM (Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) (PAA Laboratories) at 37°C and 10% (C2C12) or 5% CO2. Immortalized human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. hFOBs (1.19) were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% FCS, 2 mM l-glutamine, penicillin (100 U/ml)/streptomycin (10 µg/ml), and 0.3 mg/ml G418 (Biochrom AG) at 34°C with 5% CO2 to keep them in a proliferative state. HUVECs were a kind gift from M. Lorenz and V. Stangl (Charité Universitätsmedizin, Berlin, Germany) and cultured on gelatin-coated tissue culture ware in M199 medium supplemented with 20% FCS, 50 µg/ml endothelial cell growth supplement (Corning), 25 µg/ml heparin, 2 mM l-glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. HUVECs were used at passage 3 in all experiments. Unless stated otherwise, all cells were starved for 5 h prior to stimulation with their respective growth medium, without FCS supplement, containing 2 mM l-glutamine and penicillin/streptomycin.
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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).
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