The extracts of H. cornea and G. longissima were obtained by extraction from a lyophilized biomass with an aqueous solvent (Mili-Q® water, Millipore Corporation, Burlington, Massachusetts, United States), according to the methodology described in Álvarez-Gómez et al. [18 (link)]. From these lyophilized extracts, 20 mg was weighed and dissolved in DMEN culture medium [8 ]. Subsequently, serial dilutions were made (up to a dilution of 1:512) in order to study the effects of the concentration of algal extracts on cell viability using the MTT assay with three cell lines: RAW264.7, HGF, and HaCaT (ATCC, Manassas, WV, USA). To study the immunomodulatory capacity of the extracts, immunoassays were performed for the cytokines TNF-α and IL-6, using the same extracts of H. cornea and G. longissima but at a concentration of 0–100 μg mL−1.
Hepatocyte growth factor (hgf)
Manufactured by American Type Culture Collection
Sourced in United States
HGF is a product offered by American Type Culture Collection (ATCC) that serves as a purified protein. It is derived from recombinant human sources and is intended for use in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hacat, by American Type Culture Collection (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Mili q, by Merck Group (1 mentions)
Dermalife k medium complete kit, by Lifeline Cell Technology (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay kit, by Promega (1 mentions)
Spectramax plus, by Molecular Devices (1 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
C57bl 6, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Juli br microscope, by NanoEnTek (1 mentions)
Cho cells, by Merck Group (1 mentions)
Nucleocounter nc 200 automatic cell counter, by ChemoMetec (1 mentions)
Low serum growth kit, by American Type Culture Collection (1 mentions)
Nci h292, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
7 protocols using hepatocyte growth factor (hgf)
1
Algal Extracts Cytotoxicity and Immunomodulation
2
Gingival Cell Proliferation Assay
3
Comparative Cytotoxicity Evaluation of Fibroblasts
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For all experiments, L929 murine fibroblast cells (C57BL/6, Sigma–Aldrich, Munich, Germany) and human gingiva fibroblast cells (HGF, ATCC, Wesel, Germany) were used. L929 were cultured in minima essential medium (MEM, Gibco, Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen, Paisley, UK) and 1% penicillin/streptomycin (P/S, Gibco, Invitrogen, Paisley, UK). HGFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Paisley, UK) supplemented with 10% FBS and 1% P/S. Cells were incubated in a humified atmosphere of 95% air and 5% CO2 at 37 °C. Cells were detached at 80% confluence using 0.05% trypsin with ethylenediaminetetraacetic acid (Gibco, Invitrogen, Paisley, UK) and counted in a hemocytometer (Hecht Assistant, Sondheim vor der Rhon, Germany). Cells were seeded onto the treated or non-treated disks at a density of 0.5 × 105 /cm2, assessing cell attachment and morphology, and 1 × 105 /cm2 assessing viability and cytotoxicity.
4
Comparative Cell Viability Assessment
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Two cell types were used in the study: primary gingival fibroblasts; normal, human, adult (HGF) from ATCC (Manassas, VA, USA); and CHO cells purchased from Sigma-Aldrich (The European Collection of Authenticated Cell Cultures—ECACC). Cell cultures were carried out in an incubator at 37 °C, in a 5% CO2 atmosphere, at 95% humidity. Cells, after thawing, were cultured for at least 2 weeks prior to testing. During culturing, confluence measurement was performed using a Juli Br microscope (NanoEntek, Seoul, Republic of Korea). Cell cultures were passaged once a week with trypsin/EDTA solution. Cells for the assay were counted using a NucleoCounter® NC-200 automatic cell counter (ChemoMetec A/S, Allerod, Denmark). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) without phenol red or F-12K medium (Kaighn’s modification of Ham’s F-12 medium) supplemented with 10% fetal bovine serum (FBS), antibiotics, and L-glutamine (200 mM). Culture reagents were purchased from Biological Industries (Beit-Haemek, Israel). Detailed methodology for the assessment of cell vitality is presented by Hadzik et al. concerning experimental implant surfaces [16 (link)].
5
Culturing Human Bronchial and Gingival Cells
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Human bronchial epithelial cells (NCI-H292) (American Type Culture Collection (ATCC), were cultured at 37 °C in a 5% CO2 humidified atmosphere in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10 % Foetal Bovine Serum (FBS), 2 mM Lglutamine and 50 U/mL penicillin and 50 μg/mL streptomycin. Cells were frozen in a cell bank at passage 79. Cyropreserved H292 were recovered and cultured for a further 21 passages before disposal.
Human gingival fibroblasts (HGF; ATCC, Middlesex, UK) were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells (ATCC, Lot #6440682) were cultured in fibroblast basal media supplemented with low serum growth kit (ATCC) which includes: rh FGF b (5 ng/mL),l -glutamine (7.5 mM), Ascorbic acid (50 μg/mL), Hydrocortisone Hemisuccinate (1 μg/mL), rh Insulin (5 μg/mL) and Fetal Bovine Serum (2%). Cells were frozen in a cell bank at passage p4. Cryopreserved HGFs were recovered and cultured for a further 2–3 passages before disposal, these were either seeded into new flasks, or into 96-well plates for assays and grown to confluency before use.
Human gingival fibroblasts (HGF; ATCC, Middlesex, UK) were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells (ATCC, Lot #6440682) were cultured in fibroblast basal media supplemented with low serum growth kit (ATCC) which includes: rh FGF b (5 ng/mL),
6
HGF Cell Cultivation Protocol
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Cell cultivation followed the protocols provided by Gibco®. HGF (CRL-2014; ATCC, Manassas, VA, USA) was obtained commercially and cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Waltham, MA, USA), which was supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, Waltham, MA, USA) and incubated at 37 °C, 5% CO2. The medium was changed every 2 days. Passages 3–7 were used in the following experiments.
7
Culturing Human Gingival Fibroblasts
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The 5th passage of human gingival fibroblasts (HGF, ATCC, USA) was trypsinized in cell suspension at a cell density of 0.8 × 105/mL. The sample was seeded in 96-well plates with a cell suspension of 8 × 103 in each well and then cultured at 37°C and 95% humidity in a 5% CO2 incubator for 24 h.
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