Human dermal fibroblasts

Manufactured by Merck Group

Sourced in Poland

Human Dermal Fibroblasts (HDF) are primary cells derived from human skin tissue. They are responsible for the production of extracellular matrix components, such as collagen and elastin, which provide structural support and elasticity to the skin. HDFs play a crucial role in the skin's wound healing and repair processes.

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7 protocols using human dermal fibroblasts

Modulation of Kbhb Levels in Fibroblasts

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Bovine fibroblast cells were obtained as described previously [9] . Human Dermal Fibroblasts were purchased from Sigma (cat # 106-05A). A vial of cells was thawed, and plated at a density of 5 x 10 4 cells per 35-mm Petri dish in cell culture medium (α-MEM (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% (v/v) fetal calf serum (FCS) and 50 μg/ml gentamicin sulfate). At 24 h after plating, medium was replaced with fresh medium supplemented with:
-Bovine fibroblasts: Cultures were performed with 0 (control), 2, 4 and 6 mM BHB ((R)-(-)-3-Hydroxybutyric acid sodium salt, Sigma cat # 298360) or 5 mM Sodium Butyrate (NaBu, Sigma cat# 303410) for a period of 24 h.
-Human Dermal Fibroblasts: Cultures were performed with 0 (control), 2, 6 and 20 mM BHB for 24h.
At the end of cultures, cells were fixed with PFA 4% for immunostaining or tripsinized and recovered for histone extraction.
Regarding the experiment aimed at assessing Kbhb turnover, cells were either untreated (Control) or treated with 6 mM BHB (as described above), then washed 3 times with fresh culture medium and maintained without treatment for 0, 1, 2, 4 and 24 hours to monitor the Kbhb decay.
For the experiment involving inhibition of HDACs to increase Kbhb levels, cells were treated with 6 mM BHB, 5mM Nabu and 1 uM of TSA (Sigma cat # T8552) for 24 h.

Sangalli J.R., Nociti R.P., Del Collado M., Sampaio R.V., da Silveira J.C., Perecin F., Smith L.C., Ross P.J, & Meirelles F.V. (2022). Characterization of histone lysine β-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes. Molecular reproduction and development, 89(9).

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Establishing PARK20 Fibroblast Model

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Fibroblasts were derived directly from the skin punch biopsies of the two Italian patients carrying the p.R258Q mutation at homozygous state (Quadri et al., 2013 (link); Olgiati et al., 2014 (link)). A written informed consent was obtained from each patient. As control cells, primary adult Human Dermal Fibroblasts (HDF) were purchased from Sigma-Aldrich. PARK20 fibroblasts and HDF were grown in one ready-to-use Fibroblast Growth Medium (FGM from Sigma-Aldrich) at 37°C and 5% CO₂ in humidified atmosphere. Experiments were performed on both cell lines at similar culture passages (P5-P6). When indicated, cells were starved in Fibroblasts Basal Medium (FBM from Sigma-Aldrich), which does not contain FBS and growth factors supplement. Drug treatments were performed with 1 μM GSK2606414 (Calbiochem) or 500 nM Thapsigargin (Sigma-Aldrich) for the indicated time.

Amodio G., Moltedo O., Fasano D., Zerillo L., Oliveti M., Di Pietro P., Faraonio R., Barone P., Pellecchia M.T., De Rosa A., De Michele G., Polishchuk E., Polishchuk R., Bonifati V., Nitsch L., Pierantoni G.M., Renna M., Criscuolo C., Paladino S, & Remondelli P. (2019). PERK-Mediated Unfolded Protein Response Activation and Oxidative Stress in PARK20 Fibroblasts. Frontiers in Neuroscience, 13, 673.

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Evaluating Fibroblast Cell Culture

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Reagents: 1-Bromoheptadecafluorooctane (Sigma-Aldrich, Munchen, Germany); propidium iodide (Sigma-Aldrich), trypsin EDTA solution C (0.5%), EDTA 0.2% (10X) (Biological Industries, Kibbutz Beit HaEmek, Israel), phosphate-buffered saline (PBS) (Biomed Lublin, Lublin, Poland), MilliQ water; AQUACEL foam, nonadhesive (ConvaTec, UE) Media: Fibroblast Growth Medium (FGM) (Sigma-Aldrich, München, Germany).
Cells: Human Dermal Fibroblasts (HDF) (Sigma-Aldrich)
Bacterial cells: Escherichia coli ATCC 25922

Miłek T., Grzeczkowicz A., Lipko A., Oleksinski L., Kwiatkowska A., Strawski M., Drabik M., Stachowiak R., Goliszewski J, & Granicka L.H. (2022). A Functionalized Membrane Layer as Part of a Dressing to Aid Wound Healing. Membranes, 12(10), 936.

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Chitosan-based Antioxidant Assay

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L-lysine, propylene carbonate, NaOH, chitosan, HCl, DPPH (2,2-diphenyl-1-picrylhydrazyl) were all purchased from Sigma-Aldrich, Poznań, Poland. XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, human dermal fibroblasts (HDF), and fibroblast growth medium (FGM) were also bought from Sigma-Aldrich, Poland. Multi-hole plates (96 holes) were baught from Nest, GenoPlast, Rokocin, Poland.

Janus Ł., Piątkowski M, & Radwan-Pragłowska J. (2019). Microwave-Assisted Synthesis and Characterization of Poly(L-lysine)-Based Polymer/Carbon Quantum Dot Nanomaterials for Biomedical Purposes. Materials, 12(23), 3825.

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Chitosan-based Tissue Engineering

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Chitosan was purchased from Polaura, Poland. The iron (III) chloride, ammonium and iron (VI) sulfate, ammonia, sodium citrate, poly(vinyl alcohol), poly(ethylene glycol), poly(vinylpyrrolidone) were received from Avantor Performance Materials Poland. All compounds were characterized by analytical purity. L-Aspartic acid, fibroblast growth medium (FGM) and human dermal fibroblasts (HDF) were purchased from Sigma Aldrich, Poland. Ethanol was purchased from Avantor, Poland.

Piątkowski M., Sierakowska A., Janus Ł., & Radwan-Pragłowska J. (2020). Zaawansowane biomateriały o właściwościach półprzewodnikowych oparte chitozanie pochodzącym z grzybów. Inżynieria Mineralna, 2(1).

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Cell Culture and Transfection Protocol

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HeLa cells (kind gift from Prof. Dr. C. Azzalin, ETH Zurich, Switzerland) and human dermal fibroblasts (106–05N, Sigma-Aldrich, St. Louis, MI) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin in a 5% CO₂ atmosphere. THP1 monocytes (kind gift from Dr. H.-D. Beer, University of Zurich, Switzerland) and SW480 cells engineered for overproduction or down-regulation of HtrA1^{17 (link),18 (link)} (kind gift from Prof. Dr. M. Ehrmann, University Duisburg-Essen, Germany) were cultured in RPMI medium supplemented with 10% fetal bovine serum, 1% (v/v) penicillin/streptomycin plus G418 or puromycin, respectively, in a 5% CO₂ atmosphere. Transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions.

Sabino F., Madzharova E, & auf dem Keller U. (2020). Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival. Cell Death & Disease, 11(8), 674.

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Diverse Cell Culture Protocols Compendium

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JellaGel™ was from Jellagen (UK) and prepared as per internal specifications. All materials were used as per suppliers’ protocols. Bovine collagen type I was purchased from Gibco (Fisher Scientific, UK). Matrigel™ was purchased from Corning Inc. (USA). Incucyte® Neuroburst Orange Lentivirus reagent was from Sartorius (UK). Laminin, uridine, 5-fluro-2-deoxyuridine were purchased from Sigma. Cell lines were purchased from American Type Culture Collection (ATCC, UK): HeLa (CCL-2), MCF7 (ATCC HTB-22), MG-63 (CRL-185), A549 (CCL-185), hMSC (PCS-500-012); BIONi010–C iPSCs were from European Collection of Authenticated cell Cultures; human dermal fibroblasts were from Sigma-Aldrich (UK), and human osteoblast were from PromoCell (Germany).

Faruqui N., Williams D.S., Briones A., Kepiro I.E., Ravi J., Kwan T.O., Mearns-Spragg A, & Ryadnov M.G. (2023). Extracellular matrix type 0: From ancient collagen lineage to a versatile product pipeline – JellaGel™. Materials Today Bio, 22, 100786.

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