Geltrex

Manufactured by Ibidi

Sourced in Germany

Geltrex is a soluble basement membrane extract of extracellular matrix proteins purified from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It provides a complex mixture of extracellular matrix proteins, growth factors, and other components that support the attachment, growth, migration, and differentiation of cells in culture.

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Lab products found in correlation

Geltrex, by Thermo Fisher Scientific (3 mentions) μ slide angiogenesis plate, by Ibidi (2 mentions) 96 well μ plate, by Ibidi (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) 6 well tissue culture plate, by Corning (1 mentions) Geltrex, by Corning (1 mentions) Alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)

5 protocols using geltrex

Angiogenesis Assay using HUVEC

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A 96-well μ-plate (IBIDI) was coated with Geltrex™ diluted in fresh M199 medium (1:1 ratio) and incubated for 10 min at 4°C and for 20 min at 37°C in a humidified atmosphere containing 5% CO₂. After polymerization, HUVEC were re-suspended in complete culture medium containing GeGe3 at 20 μM and VEGF at 50 ng/ml and seeded at 10.000 cells/well. Cells were photographed with ImageXpress for 10-Hrs. The images were analyzed with ImageJ using the Angiogenesis analyzer toolset. Data were presented as the total length of the tubing segments and the network stability index, which is the ratio of total segment length and the number of isolated segments.

Meta E., Imhof B.A., Ropraz P., Fish R.J., Brullo C., Bruno O, & Sidibé A. (2017). The pyrazolyl-urea GeGe3 inhibits tumor angiogenesis and reveals dystrophia myotonica protein kinase (DMPK)1 as a novel angiogenesis target. Oncotarget, 8(64), 108195-108212.

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3D Human Skeletal Muscle Culture

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For the generation of 3D hSkM cultures, hSkM were dissociated using Trypsin (Trypsin-EDTA, 0.25%, phenol red; Life Technologies) and resuspended in Geltrex™ (Life Technologies) at a density of 3,000 hSkM per μl. Fifty ml of this viscous cell suspension were aliquoted into silicone wells (Ibidi, 80369) located inside 6-well tissue culture plates (Corning), and incubated for 30 minutes at 37°C to allow Geltrex™ gelling, at which point 4 mL of Skeletal Muscle Cell Growth Medium was added. The next day, silicone wells containing hSkM were placed into 6-well ultra-low attachment plates, and medium was changed every 2–3 days. After 7–10 days, medium was changed to Skeletal Muscle Cell Differentiation Medium to allow for differentiation of hSkM with medium changes every 2–3 days. For some experiments, including ACTA1::GCaMP6s imaging, smaller 3D hSkM were generated by resuspending 24,000 cells in 10 μL of Geltrex™ and aliquoting them in small silicone wells (Ibidi, 80409). 3D hSkM were used for assembloid generation 10 to 25 days after the switch to differentiation medium. Figures 5F and S6H show pictures of the 3D hSkM set-up.

Andersen J., Revah O., Miura Y., Thom N., Amin N.D., Kelley K.W., Singh M., Chen X., Thete M.V., Walczak E.M., Vogel H., Fan H.C, & Paşca S.P. (2020). Generation of Functional Human 3D Cortico-Motor Assembloids. Cell, 183(7), 1913-1929.e26.

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Immunofluorescence Analysis of iPSC-RPE Cells

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Immunofluorescence was performed in 12-well Geltrex-coated plates or 8-well Ibidi µ-slides (Ibidi GmbH, Planegg/Martinsried, Germany) with confluent iPSC-RPE cells. Cells were rinsed 3 times with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 1% Triton X-100 for 15 min, and blocked with FBS 20% + 0.1% Triton X-100 for 1 h. We used the following antibodies: ZO-1 (1A12) mouse monoclonal antibody (Invitrogen, Waltham, MA, USA), BEST1 (E6-6) mouse monoclonal (Invitrogen), RPE65 (MA1-16578) mouse monoclonal (Invitrogen), and MITF (Ab20663) rabbit polyclonal (Abcam, Cambridge, UK). Primary antibody incubation was performed O/N with 1% FBS at 4 °C. Primary antibodies were tagged with an anti-mouse Alexa-488 secondary antibody (Invitrogen) or an anti-rabbit Alexa-586 secondary antibody (Invitrogen) for 1h at RT. Cell nuclei were stained with DAPI (Thermofisher Scientific) for 10 min.
Images were obtained either with a ZEISS Axio Vert.A1 or a Zeiss LSM980 confocal microscope (Carl Zeiss Sports Optics, Jena, Germany) and processed and quantified by ImageJ software.

Navinés-Ferrer A., Ruiz-Nogales S., Navarro R, & Pomares E. (2022). Impaired Bestrophin Channel Activity in an iPSC-RPE Model of Best Vitelliform Macular Dystrophy (BVMD) from an Early Onset Patient Carrying the P77S Dominant Mutation. International Journal of Molecular Sciences, 23(13), 7432.

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Quantifying Angiogenic Network Formation

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Tube formation assay was used as a model for angiogenesis. Geltrex™ (Life Technologies, Grand Island, NY, USA) was thawed overnight at 4°C. Using cold pipette tips, 10 μl/well of Geltrex™ was added to a μ-slide angiogenesis plate (ibidi GmBH, Germany). The Geltrex™ solidified into a thin layer after incubation at 37°C for 1 hour. RF/6A endothelial cells were pre-treated with drugs or endogenous ligands for 24 hours (Supplementary Table 1 and 2), trypsinized and then plated onto the Geltrex™-coated wells (12,000 cells/well). Network formation was examined after 3 hours, using an inverted phase contrast microscope, and quantified as total tube length formed by endothelial cells normalized to DMSO control, using ImageJ. Four fields of view/experiment were examined, in a total of three biological replicates.

Choudhary M., Safe S, & Malek G. (2018). Suppression of aberrant choroidal neovascularization through activation of the aryl hydrocarbon receptor. Biochimica et biophysica acta, 1864(5 Pt A), 1583-1595.

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Angiogenesis Assay for Primary Endothelial Cells

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Tube formation assay was used as an in vitro angiogenesis model to validate 1°CEC function as previously described [36 (link)]. Briefly, Geltrex™ (Life Technologies, Grand Island, NY, USA) was thawed overnight at 4 °C. Using cold pipette tips, 10 μL/well of Geltrex™ was added to a μ-slide angiogenesis plate (ibidi GmBH, Gräfelfing, Germany). The Geltrex™ solidified into a thin layer after incubation at 37 °C for 1 h. 1°CECs were plated at a density of 12,000 cells/well and incubated at 37 °C, in 5% CO₂. After 3 h, network formation was imaged using an inverted phase-contrast microscope (Zeiss, Axio Observerer.D1, Whiteplains, NY, USA).

Peavey J., Parmar V.M, & Malek G. (2022). Nuclear Receptor Atlases of Choroidal Tissues Reveal Candidate Receptors Associated with Age-Related Macular Degeneration. Cells, 11(15), 2386.

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