Fibroblast growth medium 3

Manufactured by PromoCell

Sourced in United States

Fibroblast Growth Medium 3 is a specialized cell culture medium designed to support the growth and maintenance of fibroblast cells. It provides the necessary nutrients and growth factors required for the cultivation of fibroblasts in an in vitro setting.

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Lab products found in correlation

Penicillin streptomycin, by Charles River Laboratories (1 mentions) Basic fibroblast growth factor (bfgf), by PromoCell (1 mentions) Insulin, by PromoCell (1 mentions) Penicillin streptomycin, by PromoCell (1 mentions) A10042, by Thermo Fisher Scientific (1 mentions) A21202, by Thermo Fisher Scientific (1 mentions) A21447, by Thermo Fisher Scientific (1 mentions) Ab32575, by Abcam (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Huvecs, by Lonza (1 mentions) Endothelial cell growth medium 2, by PromoCell (1 mentions) Endothelial cell growth medium mv2, by PromoCell (1 mentions) Ab152163, by Abcam (1 mentions) Nhcf 5, by Lonza (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) Myocyte growth media, by PromoCell (1 mentions)

Spelling variants of this product

Fibroblast Growth Medium (22 citations) Fibroblast Growth Medium −3 (FGM-3) (2 citations)

7 protocols using fibroblast growth medium 3

PDX Cell Line Culture Protocol

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PDX-derived cell lines were supplied by Charles River from Charles River’s Cancer Model Database (https://compendium.criver.com/ (accessed on 18 November 2021)) and were grown at 37 °C in a humidified atmosphere with 5% CO₂ in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 U/mL Penicillin-Streptomycin. PDX cells were used within 10 passages and are listed in Table 1 [16 (link)].
Human dermal fibroblasts (HDF, ScienCell Research Laboratories, Carlsbad, CA, USA) were grown at 37 °C in a humidified atmosphere with 5% CO₂ in Fibroblast growth medium 3 supplemented with 10% fetal bovine serum, 100 U/mL Penicillin-Streptomycin, 1 ng/mL basic fibroblast growth factor and 5 μg/mL insulin (PromoCell, Heidelberg, Germany).

Xue B., Schüler J., Harrod C.M., Lashuk K., Bomya Z, & Hribar K.C. (2023). A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies. Cells, 12(8), 1145.

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Cardiac Fibroblast Expansion and Characterization

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Expansion and maintenance of CFs with Fibroblast Growth Medium 3 (Promocell, Heidelberg, DE) and 0.25% Trypsin allowed for expansion through at least 16 passages. CFs of passage 8 or below were cultured in either Fibroblast Growth Medium 3 or RPMI1640 + 10% FBS and 250 μM of ascorbic acid for 3 days and stained for αSMA (ab32575, Abcam, 1:500) as an activation marker, TE-7 (NBP2–50082, Novus Bio, 1:100) and PDGFRα (AF-307-NA, RnD Systems, 1:250), to confirm identity, and fibronectin EDA (NBP1–51723, Novus Bio, 1:200) to confirm matrix competency. WT1 (R&D Systems, Cat. AF5729, Minneapolis, MN) and vimentin (Cell Signaling Technology, Cat. 5741, Danvers, MA) were used as epicardial and post-epithelial to mesenchymal transition markers. TCF21 (PA5–53031, ThermoFisher, 1:100) was used to confirm epicardial lineage and requisite CF transcription factor expression. Samples were blocked in 10% donkey serum, 0.3 M glycine, and 1% bovine serum albumin for 1 h, permeabilized in blocking buffer with 0.1% Triton X-100 for 20 min, stained with primary antibodies for 2 h, and then incubated with secondary antibodies (A21202, A10042, and A21447, Invitrogen) for another two hours. Nuclei were stained with DAPI for 15 min at 1:10,000 dilution in DI water, and three washes were performed between each incubation for 5 min each.

Whitehead A.J., Hocker J.D., Ren B, & Engler A.J. (2021). Improved epicardial cardiac fibroblast generation from iPSCs. Journal of molecular and cellular cardiology, 164, 58-68.

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Chikungunya Virus Infection of Primary Cardiac Cells

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Human primary cardiac cells were seeded into 96-well plates flat transparent black plates (Corning, 07200588) at a density of 18,000 cells/well. After 24 h cells were stimulated with the indicated concentrations of IFNα A/D (Millipore Sigma, #I4401) or IFNβ (Millipore Sigma, #IF014) for 24 h. Cells pretreated with IFN or mock-treated were infected with CHIKV-ZsGreen at an MOI of 0.25 or mock-infected with DMEM for 1 h at 37 °C. After 1 h, the inoculum was removed, monolayers were washed once with cold PBS, and 100 μl of Fibroblast Growth Medium 3 (PromoCell, C-23025) was added to the cells. Cells were incubated at 37 °C with 5% CO₂ for 24 h. Cells were fixed with 10% formalin for 1 h, and stained for microscopy (see below).

Noval M.G., Spector S.N., Bartnicki E., Izzo F., Narula N., Yeung S.T., Damani-Yokota P., Dewan M.Z., Mezzano V., Rodriguez-Rodriguez B.A., Loomis C., Khanna K.M, & Stapleford K.A. (2023). MAVS signaling is required for preventing persistent chikungunya heart infection and chronic vascular tissue inflammation. Nature Communications, 14, 4668.

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Vascular Cell Isolation and Culture

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HUVECs were purchased from Lonza (from pooled donors) and cultured in Endothelial Cell Growth Medium 2 (EGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human pericytes (from placenta) were purchased from PromoCell and cultured in Pericytes Growth Medium 2 (PGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human cardiac endothelial cells and fibroblasts were purchased from PromoCell and cultured in Endothelial Cell Growth Medium MV 2 and Fibroblast Growth Medium 3 respectively (PromoCell). Endothelial cells were cultured on 0.1% gelatin treated T75 flasks. We note that although placental pericytes and HUVECs are not normally found in the cardiac microvasculature, they constitute a reliable source of relevant cells allowing the reproducible assembly of complex in vitro models.

Di Cio S., Marhuenda E., Haddrick M, & Gautrot J.E. (2024). Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics. Scientific Reports, 14, 3370.

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Culturing Human Cardiac Fibroblasts

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To validate our findings across species, we cultured human cardiac fibroblasts isolated from ventricles of adult heart (HCF, PromoCell # C-12377) in fibroblast growth medium 3 (PromoCell) until P5. Confluent cells were starved overnight in SFM and incubated with p1159 as described above (Table 1). After incubation, cells were rinsed and either fixed for immunofluorescence or lysed for protein and/or RNA extraction. Protein lysates were obtained with 1x ice cold lysis buffer with 1x protease inhibitors (Abcam, ab152163), according to manufacturer’s instructions and quantified for immunoblotting.

Shigenaga M.K., Lee H.H., Blount B.C., Christen S., Shigeno E.T., Yip H, & Ames B.N. (1997). Inflammation and NOx-induced nitration: Assay for 3-nitrotyrosine by HPLC with electrochemical detection. Proceedings of the National Academy of Sciences of the United States of America, 94(7), 3211-3216.

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Cardiac Fibroblast Culture Protocol

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Primary human ventricular cardiac fibroblasts (NHCF-V; Lonza, cat. no. CC-2904) were cultured according to the manufacturer’s protocol with Fibroblast Growth Medium 3 (PromoCell, cat. no. C-23130). Fibroblasts were used for engineering tissues between passage 3 and passage 5. Cells were generated from healthy donors with no history of SLE or cardiovascular disease.

Fleischer S., Nash T.R., Tamargo M.A., Lock R.I., Venturini G., Morsink M., Li V., Lamberti M.J., Graney P.L., Liberman M., Kim Y., Zhuang R.Z., Whitehead J., Friedman R.A., Soni R.K., Seidman J.G., Seidman C.E., Geraldino-Pardilla L., Winchester R, & Vunjak-Novakovic G. (2024). An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury. bioRxiv.

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Culturing Diverse Cardiac Cell Types

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Cells were maintained at 37 °C in 5% CO₂. Baby hamster kidney (BHK-21, ATCC CCL-10) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% nonessential amino acids (NEAA, Gibco). Vero cells (ATCC CCL-81) were grown in DMEM with 10% newborn calf serum (NBCS; Gibco). Human primary cardiac cells were obtained from PromoCell, used up to passage 10, and grown following the company recommendations. Human Cardiac Fibroblast (C-12375), human Cardiac Microvascular Endothelial Cells (C-12285), and human Cardiac Myocytes (C-12810) were grown in Fibroblast Growth Medium 3 (PromoCell, C-23025), Endothelial Cell Growth Media MV (PromoCell, C-22020), and Myocyte Growth Media (PromoCell, C-22070), supplemented with 1% Penicillin-Streptomycin (Cellgro, 30-002-CI), respectively. All the cell types were confirmed free of mycoplasma using Lookout Mycoplasma PCR detection kit (Sigma-Aldrich).

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