F0895

Manufactured by Merck Group

Sourced in United States, United Kingdom, Switzerland

The F0895 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory applications. The core function of this product is to provide a reliable and efficient tool for laboratory tasks, but a detailed description of its intended use is not available.

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Lab products found in correlation

42 protocols using f0895

Culturing and Stimulating Human Cardiac Fibroblasts

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Primary human cardiac fibroblasts (HCF) (#C-12375, Promocell) or immortalized human cardiac fibroblasts (iHCF) (#P10453-IM, Innoprot) were used. HCF were cultured in Fibroblast Growth Medium 3 (#C-23130, Promocell) according to the manufacturer’s protocol. HCF were seeded at a density of 5000 cells/cm² in 6-well, 12-well plates and 8-well µ-Slides (#80826, IBIDI) coated with human fibronectin (#F0895, Sigma Aldrich, 0.1% solution; 1:1000 in water) for indirect co-culture, RNA extraction and qRT-PCR analysis. Immortalized iHCF were cultured in Dulbecco modified Eagles medium (DMEM) (#11965092, Thermo Fisher Scientific) supplemented with 10 % FBS (#16000044, Thermo Fisher Scientific) and 50 U/mL Penicillin and Streptomycin according to the manufacturer’s protocol. HCF were seeded at a density of 17 500 cells/cm² in fibronectin-coated (#F0895, Sigma Aldrich, 0.1% solution; 1:1000 in water) 12-well plates and 8-well µ-Slides for indirect co-culture, RNA extraction and qRT-PCR analysis.
For stimulation with recombinant human HB-EGF (#100-47, PeproTech), fibroblasts were seeded in the respective densities one day prior to the stimulation. One day after stimulation, medium was replaced with fresh medium containing 100 ng/ml HB-EGF for 48 h.

Shumliakivska M., Luxán G., Hemmerling I., Scheller M., Li X., Müller-Tidow C., Schuhmacher B., Sun Z., Dendorfer A., Debes A., Glaser S.F., Muhly-Reinholz M., Kirschbaum K., Hoffmann J., Nagel E., Puntmann V.O., Cremer S., Leuschner F., Abplanalp W.T., John D., Zeiher A.M, & Dimmeler S. (2024). DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts. Nature Communications, 15, 606.

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Culturing HUVEC and HeLa Cells

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Human umbilical vein endothelial cells (HUVEC) were kindly provided by Sérgio Dias (Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal) and cultured in EBM2-supplemented medium (Lonza) with 5% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), penicillin (100 U/mL)–streptomycin (100 µg/mL) solution (Hyclone, Thermo Scientific) and 2 mM l-glutamine (Hyclone, Thermo Scientific), on 0.2% (w/v) gelatin – (G1890, Sigma-Aldrich), 10 µg/mL laminin – (L2020, Sigma-Aldrich) or 10 µg/mL fibronectin – (F0895, Sigma-Aldrich) coated plates. Passages used were between 3 and 12.
HeLa cervical carcinoma cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Hyclone, Thermo Scientific) supplemented with 10% (v/v) FBS, penicillin (100 U/mL)–streptomycin (100 µg/mL) solution (Hyclone, Thermo Scientific) and 2 mM l-glutamine (Hyclone, Thermo Scientific), on uncoated or 0.2% (w/v) gelatin- (G1890, Sigma-Aldrich), 10 µg/mL laminin – (L2020, Sigma-Aldrich), 10 µg/mL fibronectin – (F0895, Sigma-Aldrich) or 120 µg/mL collagen I – (#3440-100-01, R&D Systems) coated plates.

Bagulho A., Vilas-Boas F., Pena A., Peneda C., Santos F.C., Jerónimo A., de Almeida R.F, & Real C. (2015). The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells. Redox Biology, 6, 454-460.

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Exploring Glycated Albumin and Chemokine Effects on Fibronectin Production

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30 mM D-glucose was used as high glucose concentration for the following experiments, with Mannitol used as the osmotic control (26 mM +4 mM of D-glucose). Glycated albumin (A8301, Sigma, Gillingham, UK) was used at a concentration of 500 μg/ml +4 mM D-glucose, with physiological 4 mM D-glucose used as its control. The Fn ELISA was carried out using rabbit anti-human Fn polyclonal Ab for capture (F3648, Sigma, Gillingham, UK) and biotinylated murine anti-human Fn monoclonal Ab (F7387, Sigma, Gillingham, UK) for detection. Fn derived from human plasma was used as the antigen for the standard curve (range 1.95-2000 pg/ml) (F0895, Sigma, Gillingham, UK).
Recombinant human CCL18 (394-PA, R&D systems, Abingdon, UK) and MCP-1 (279-MC, R&D systems, Abingdon, UK) were reconstituted according to the manufacturer’s instructions. Following dose response experiments, HK-2 cells were stimulated with recombinant CCL18 or rMCP-1 at a concentration of 20 ng/ml for 48 h. Fn levels were subsequently measured from the cell supernatant using ELISA.

Montero R.M., Bhangal G., Pusey C.D., Frankel A.H, & Tam F.W. (2016). CCL18 synergises with high concentrations of glucose in stimulating fibronectin production in human renal tubuloepithelial cells. BMC Nephrology, 17, 139.

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Quantifying Spore Adhesion on ECM Coated Plates

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96-well flat bottom plates were coated with 0–5
μg/cm² laminin (Sigma Aldrich Product: AG56P) or
fibronectin (Sigma-Aldrich Product: F0895) for 2 h at room temperature.
Thereafter, plates were washed with E3 medium and 1000 spores of R.
arrhizus (clinical isolate #749, CotH RNAi, or Control RNAi),
suspended in 100 μL E3 medium, were added to each well. After
overnight incubation (16 h) on the nutator at 28°C, wells were
carefully washed twice with fresh E3 medium to remove non-adherent spores.
The number of remaining (adherent) spores was then quantified using the
IncuCyte ZOOM microscopy platform and its Basic Analyzer module in
combination with previously developed detection algorithms (Wurster et al., 2019 (link)). Four wells were tested for
each extracellular matrix compound and concentration.

Wurster S., Ruiz O.E., Samms K.M., Tatara A.M., Albert N.D., Kahan P.H., Nguyen A.T., Mikos A.G., Kontoyiannis D.P, & Eisenhoffer G.T. (2021). EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion. Cell reports, 34(12), 108896.

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Quantifying Plasma Protein Adsorption

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Bradford assay was used to study the time-course of blood plasma proteins and of pure FBN adsorption to chitosan with or without aptamers.
To this purpose, discs were soaked for 1 h in 200 µL of PBS supplemented with 2% of human serum (Pooled Normal Human Serum, Innovative Research, Peary Court, FL, USA) either of pure FBN (F0895, Sigma-Aldrich) at a concentration comparable with that present in a 2% human serum solution (0.02 mg/mL). Protein amount in supernatants was quantitated after 5, 15, 30, 45 and 60 min through Bradford assay (BIO-RAD Protein Assay, BIO-RAD, Hercules, CA, USA) according to the manufacturer’s recommendations. Ten-µl aliquots were collected at each time point, mixed with 200 µL of Bradford Working Solution and incubated at 37 °C for 2 min prior to absorbance assessment at 620 nm with a micro plate photometer (Multiskan™ FC, Thermo Fisher Scientific, Waltham, MA, USA). The number of proteins deposited on the discs was finally calculated by subtracting the residual concentration in supernatant from the initial one.

Parisi L., Toffoli A., Bianchi M.G., Bergonzi C., Bianchera A., Bettini R., Elviri L, & Macaluso G.M. (2019). Functional Fibronectin Adsorption on Aptamer-Doped Chitosan Modulates Cell Morphology by Integrin-Mediated Pathway. Materials, 12(5), 812.

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Endothelial Cell Migration Assay

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Upon reaching confluency HBMECs were starved for 5 h in HBMECs starvation medium, then trypsinized and plated at 4 × 10⁴cells/insert in a Transwell Permeable Support (6.5 mm Insert, 24-well plate, 3.0 μm polycarbonate membrane, tissue culture-treated polystyrene, Costar (Ref: 3415)) with control CM, control CM+50 ng ml⁻¹ VEGF, MN-CM or IN-CM (all with or without adding 1 μg ml⁻¹ recombinant mouse Flt1/Fc) in the lower compartment. Inserts were coated before with 10 μg ml⁻¹ fibronectin (F0895, Sigma-Aldrich). After 20 h cells were fixed and nuclei were stained. Migration of endothelial cells on the other side of the filter was calculated by counting the number of nuclei per field of view. Five field of views were counted per condition. Quantification was done blind to the experimental condition.

Himmels P., Paredes I., Adler H., Karakatsani A., Luck R., Marti H.H., Ermakova O., Rempel E., Stoeckli E.T, & Ruiz de Almodóvar C. (2017). Motor neurons control blood vessel patterning in the developing spinal cord. Nature Communications, 8, 14583.

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Live-Cell Microscopy of Wnt Signaling

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The TIRF experiments were done on a Nikon Ti motorized inverted microscope with Perfect Focus System, 491-nm laser (50 mW max power) with 50-ms exposure time. Images or movies were acquired with MetaMorph software. The microscope has temperature and CO₂ level control to minimize the environment perturbation on cells.
The cells were either grown on γ-irradiated 35-mm glass bottom dishes (MatTek P35G-0.170-14-C) or on high-precision microscope cover glasses (Marienfeld 0117650 lot. 33825 819). The glass was coated with 10 μg/mL fibronectin (Sigma-Aldrich F0895) in PBS for 10 min before cells were plated. A total of 100 nM LGK974 (Caymanchem 14072) was added to the cell culture medium when the cells were plated to decrease the autocrine Wnt signaling. The cells were cultured in normal DMEM and switched into FluoroBrite (Thermo Fisher A1896701) with 10% FBS and 100 nM LGK974 before imaging. The imaging experiments were normally done the next day after the cells were plated.

Ma W., Chen M., Kang H., Steinhart Z., Angers S., He X, & Kirschner M.W. (2020). Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation. Proceedings of the National Academy of Sciences of the United States of America, 117(28), 16690-16701.

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HUVEC Cell Viability Assay

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For detection of live cells, nuclei of HUVECs were stained with NucBlue^® Live (live DAPI; R37605, Life Technologies, Eugene, OR, USA) for 20 mins according to the manufacturer’s instructions. The cells were seeded in fibronectin (F0895, Sigma-Aldrich)-coated six-well plates at a density of 5 × 10⁴ cells/well with either DMSO or KCH-1521-containing culture medium. After 10 mins of attachment, the unattached cells were washed twice with 1× PBS and fixed with 2% paraformaldehyde (PFA; P6148, Sigma-Aldrich). All images were taken immediately using an upright fluorescence microscope (DMI 3000B, Leica Microsystems, Wetzlar, Germany) and the live adherent cells were counted in five different fields per well.

Lim I.R., Joo H.J., Jeong M., Kim J.H., Choi S.C., Kim C., Jung J.W, & Hong S.J. (2017). Talin Modulation by a Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells. International Journal of Molecular Sciences, 18(1), 221.

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Mechanical Properties of Coated Scaffolds

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The LOCS was prepared as previously described.⁵³ 10% poly‐l‐ornithine (P4957, Millipore) and 1% fibronectin (F0895, Sigma‐Aldrich) were coated on the LOCS to obtain P/F‐LOCS. A mechanical‐testing machine (Shimadzu AGS‐X, Japan) was used to test the mechanical properties of the LOCS and P/F‐LOCS. Stretching strain data at various stretching stresses were recorded (n = 6 samples). The scaffolds with or without cells were visualized with a scanning electron microscope (EVO LS 10, Germany) after desiccation with a supercritical point dryer and sputter‐coating with gold (n = 6 samples).

Jin C., Wu Y., Zhang H., Xu B., Liu W., Ji C., Li P., Chen Z., Chen B., Li J., Wu X., Jiang P., Hu Y., Xiao Z., Zhao Y, & Dai J. (2022). Spinal cord tissue engineering using human primary neural progenitor cells and astrocytes. Bioengineering & Translational Medicine, 8(2), e10448.

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Astrocyte Differentiation and Characterization

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Control isogenic and DS iPSCs were differentiated into neural progenitors and cultured similar to neural differentiation with the following modifications. Astrospheres were then plated at a concentration of 500,000 cells/mL in 12 mL of media in T75 flasks and allowed to adhere to fibronectin-coated dishes (Sigma; F0895). After confluent, astrospheres were dissociated into single cells and cultured in an optimized commercial medium for human primary astrocytes (ScienCell Research Laboratories; 1801). HAs were from ScienCell Research Laboratories (1800). We performed karyotype analysis on DS1-, DS4-, and DS2U-derived astroglia, prior to and after the Ca²⁺ experiments (Cell Line Genetics). The cell size was analyzed by randomly selecting 5 cells from 3 bright field images (ImageJ).

Mizuno G.O., Wang Y., Shi G., Wang Y., Sun J., Papadopoulos S., Broussard G.J., Unger E.K., Deng W., Weick J., Bhattacharyya A., Chen C.Y., Yu G., Looger L.L, & Tian L. (2018). Aberrant Calcium Signaling in Astrocytes Inhibits Neuronal Excitability in a Human Down Syndrome Stem Cell Model. Cell reports, 24(2), 355-365.

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