Growth factor reduced matrigel

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Growth factor-reduced Matrigel is a complex extracellular matrix (ECM) preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is a soluble basement membrane extract that solidifies to form a gel-like material when warmed to physiological temperature.

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Matrigel (17210 citations)

821 protocols using growth factor reduced matrigel

Xenotransplantation of Undifferentiated Spermatogonia

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Two groups were created: Group 1 (control): 10⁷ cells in 100μl of Matrigel reduced growth factors (BD Sciences, Bedford, United States) and Group 2: 10⁷ cells containing 25% of undifferentiated spermatogonia enriched by stirred suspension culture (approximately 50% of the cell pellet content derived from the stirred suspension bioreactor) in 100μl of Matrigel reduced growth factors. Cell pellets weighed was 32±2.3mg.
Cell grafting was performed essentially as described (Dores & Dobrinski, 2014 ). Immunodeficient recipient mice (9 NU/NCr, Charles River, Wilmington, United States) were castrated and two cell pellets from each group were randomly assigned to one of four injection sites located under the dorsal skin, spaced between the shoulder and the rump. The experiment was performed in three replicates with different pools of testicular cells, to minimize any influence from the cell suspension, surgical procedure or batch of mice used on the observed results.
The care and use of research animals were approved by the Institutional Animal Care and Use Committee of the University of Calgary.

Dores C., Rancourt D, & Dobrinski I. (2015). Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis. Andrology, 3(3), 590-597.

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Exosome-Mediated Cell Migration and Angiogenesis

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BD Falcon™ Cell Culture Inserts with 8 μm pore size were used for migration assay and the same inserts coated with 10% growth factor reduced Matrigel were used for invasion assay (BD Biosciences). Cells were seeded onto the top chamber of a transwell insert in 300 μl exosome-free medium and 600 μl of serum-free medium was added into the bottom chamber in a 24-well plate. Exosomes were added four hours later onto the top chamber, at 2 concentrations as detailed above (1X and 10X). Cells incubated in exosome-depleted medium were used as a control. The inserts were fixed after 24–72 hours, stained with hematoxylin and eosin (H&E stain), mounted and coverslipped. Slides were air-dried, sealed, photographed and the migrating/invading cells counted using ImageJ software.
The formation of capillary-like structures was assessed in a 96-well plate using growth factor-reduced Matrigel (BD Biosciences). HUVECs (1.5 × 10⁴cells/well) were plated on top of Matrigel in endothelial basal medium containing 1% FBS. Exosomes were added at a concentration of 1X and 10X, and exosome-free medium was used as control. Cells were incubated for 2 hours and then evaluated by phase-contrast microscopy and photographed. The number of nodes (defined as the intersection of 3 or more branches) formed in each condition was counted using the ImageJ software.

Ghayad S.E., Rammal G., Ghamloush F., Basma H., Nasr R., Diab-Assaf M., Chelala C, & Saab R. (2016). Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts. Scientific Reports, 6, 37088.

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Matrigel Tube Formation Assay

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Matrigel tube formation assay using growth factor-reduced Matrigel (BD Biosciences) was performed. HMVECs were seeded in Labtek chamber slides on growth factor reduced Matrigel (Becton Dickinson, Bedford, MA, USA) at a density of 1.6 × 10⁴ cells per chamber. The test substances used were rhuId1 (1 and 10 nM), bFGF (60 nM, R&D Systems, Minneapolis, MN, USA; positive control) and PBS (negative control). The treated HMVECs (1.8 × 10⁴ cells/400 μl) were plated on Matrigel in the presence of Id1, bFGF or PBS for six hours at 37°C. Photographs (100×) were taken and tubes were counted by a blinded observer. Tubes were defined as elongated connecting branches between two identifiable HMVECs. SFs were diluted 1:100 with PBS. Matrigel tube formation assay was performed using SFs and PBS. Photographs (100×) were taken and tubes were counted by a blinded observer.

Isozaki T., Amin M.A., Arbab A.S., Koch A.E., Ha C.M., Edhayan G., Haines GK I.I.I, & Ruth J.H. (2014). Inhibitor of DNA binding 1 as a secreted angiogenic transcription factor in rheumatoid arthritis. Arthritis Research & Therapy, 16(2), R68.

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Inducible TMEFF2 Expression in TRAMPC2 Cells

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The TRAMPC2 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM supplemented with 0.005 mg/ml bovine insulin and 10 nM dehydroisoandrosterone, 90%; fetal bovine serum, 5%; Nu-Serum IV, 5% as recommended. For matrigel growth, cells were trypsinized, mixed 1:1 with culture medium containing 4% growth factor reduced Matrigel (BD Biosciences, San Jose, CA) and added to the wells of a 96-well plate coated with 35 μl of growth factor reduced Matrigel at low concentration (4 mg/ml) per well as described by Debnath et al. (32 (link)). Culture medium containing 2% Matrigel was replaced every 3 days. TMEFF2 full-length expression construct was as previously described (25 (link)). Development of a system for inducible expression of TMEFF2 (TMEFF2i) in TRAMPC2 cells was achieved using the Clontech’s Tet-On Advanced system as described before for other cell lines (25 (link)). To inducibly express TMEFF2, cultures were grown in the presence of doxycycline (250 ng/ml; SIGMA, St. Louis, MO). The probasin-TMEFF2 expression construct was generated by inserting the human full length TMEFF2 cDNA into the pTg1 vector downstream from the ARR₂PB minimal rat probasin promoter and intervened by an intron sequence.

Corbin J., Overcash R., Wren J., Coburn A., Tipton G., Ezzell J., McNaughton K., Fung K., Kosanke S, & Ruiz-Echevarria M. (2015). ANALYSIS OF TMEFF2 ALLOGRAFTS AND TRANSGENIC MOUSE MODELS REVEALS ROLES IN PROSTATE REGENERATION AND CANCER. The Prostate, 76(1), 97-113.

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Acini Formation Assay Protocol

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8-well chamber slides (BD Biosciences) were coated with 30μl of growth factor reduced matrigel (BD Biosciences). 5000 cells were plated per well in assay media containing 2% growth factor reduced matrigel (BD Biosciences) and 5 ng/ml EGF. The medium was replaced every 3 days. On 12th day images were taken and using Olympus Cell^F software diameter was measured and recorded for 100–150 acini for each stable cell line. Statistical analysis was performed with Prism software.

Kulkarni M., Tan T.Z., Syed Sulaiman N.B., Lamar J.M., Bansal P., Cui J., Qiao Y, & Ito Y. (2018). RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer. Oncotarget, 9(18), 14175-14192.

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Cell Invasion and Tube Formation Assays

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For invasion assays, transwell cell culture inserts (8-μm pore size, 24 wells, BD Biosciences) were coated with 1 mg/ml of growth factor-reduced Matrigel (BD Biosciences). Treated cells (1 × 10⁵ cells/insert) in medium supplemented with 0.1% FBS were incubated for 48 h against a gradient of 10% FBS. Non-invading cells were removed with a cotton swab from the upper side of the membrane. Cells that penetrated the membrane were fixed with cold methanol and stained with crystal violet (0.5%, Sigma) for 30 min and subsequently washed thoroughly with tap water.
For endothelial-like tube formation assays, individual wells of a 96-well plate were each coated with 30 μl growth factor-reduced Matrigel (BD Biosciences, 5 mg/ml). Coated wells were seeded with 4 × 10⁴ HTR-8/SVneo cells suspended in 100 μl DMEM/F12 medium supplemented with 0.1% FBS and incubated at 37°C for 24 h. Digital images were taken with a light microscope and data was analyzed with Image-J software.

Yi Y., Zhu H., Klausen C., Chang H.M., Inkster A.M., Terry J, & Leung P.C. (2021). Dysregulated BMP2 in the Placenta May Contribute to Early-Onset Preeclampsia by Regulating Human Trophoblast Expression of Extracellular Matrix and Adhesion Molecules. Frontiers in Cell and Developmental Biology, 9, 768669.

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Matrigel-Based Morphogenesis Assay

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Morphogenesis assays were conducted in the growth factor reduced Matrigel (BD Biosciences) as previously described [54 (link)]. Cells were seeded in 8-well chamber slides containing a solid base layer of growth factor reduced Matrigel (BD Biosciences). Photographs were taken with a Nikon SMZ 1500 stereoscopic zoom microscope.

Cravero K., Pantone M.V., Shin D.H., Bergman R., Cochran R., Chu D., Zabransky D.J., Karthikeyan S., Waters I.G., Hunter N., Rosen D.M., Kyker-Snowman K., Dalton W.B., Button B., Shinn D., Wong H.Y., Donaldson J., Hurley P.J., Croessmann S, & Park B.H. (2022). NOTCH1 PEST domain variants are responsive to standard of care treatments despite distinct transformative properties in a breast cancer model. Oncotarget, 13, 373-386.

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Xenograft Tumor Study in SCID Mice

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Xenograft tumor studies were conducted as previously described.
[ 24 ] Briefly, severe combined immunodeficiency (SCID) mice (4-6 weeks old) were purchased from the Shanghai SLAC laboratory animal company. Animals were given time to adapt to the sterile-and pathogen-free environment with food and water ad libitum. CD133 + CD34 -and CD133 -CD34 + cells were isolated according to the manufacturer's manual. Viable cells of an indicated number in 50 μl of sterile PBS suspensions were mixed with 100 μl of reduced growth factor Matrigel (BD Biosciences, USA) and injected bilaterally and subcutaneously into SCID mice. On day 42 postcell injection, animals were euthanized by cervical dislocation, and tumors were removed and weighed. H1650 PLKO.1 and H1650 miR-27a cells were harvested in the exponential growth phase using a PBS/ethylenediaminetetraacetic acid solution and washed. Viable cells of an indicated number in 50 μl of sterile PBS suspension were mixed with 100 μl reduced growth factor Matrigel (BD Biosciences, USA) and injected subcutaneously into the SCID mice. On day 21 postcell injection, tumor size was measured with a digital caliper and calculated using the formula: 4/3πLS2 (L = larger radius, S = smaller radius).

Luo W., Zhang D., Ma S., Wang C., Zhang Q., Wang H., He K, & Liu Z. (2018). miR-27a is highly expressed in H1650 cancer stem cells and regulates proliferation, migration, and invasion. Journal of cancer research and therapeutics, 14(Supplement).

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Angiogenic Potential of MMECs

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MMECs treated as above were plated on Matrigel reduced growth factor (Becton Dickinson Biosciences, Bedford, MA) coated 48-well plates in serum-free medium (SFM) or in presence of anti EphA3 or isotype Ab after 18h, the skeletonization of the mesh was followed by measurement of mesh areas and vessel length in three randomly-chosen fields with the EVOS microscope at ×200.

Caivano A., Rocca F.L., Laurenzana I., Annese T., Tamma R., Famigliari U., Simeon V., Trino S., Luca L.D., Villani O., Berardi S., Basile A., Vacca A., Saglio G., Vecchio L.D., Musto P, & Cilloni D. (2017). Epha3 acts as proangiogenic factor in multiple myeloma. Oncotarget, 8(21), 34298-34309.

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Quantifying Cellular Adhesion and Angiogenesis

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Adhesion: One×10 Images were captured using a Nikon Eclipse TE2000-5 microscope. Four selected field of images were captured in each sample, and the wound areas were estimated by Nikon NIS-Elements computer software.
Angiogenesis on Matrigel: MMECs treated as above were plated on Matrigel reduced growth factor (Becton Dickinson Biosciences, Bedford, MA) coated 48-well plates in serum-free medium (SFM) or in presence of anti EphA3 or isotype Ab. After 18h, the skeletonization of the mesh was followed by measurement of mesh areas and vessel length in three randomly-chosen fields with the EVOS microscope at X200.

La Rocca F., Airoldi I., Di Carlo E., Marotta P., Falco G., Simeon V., Laurenzana I., Trino S., De Luca L., Todoerti K., Villani O., Lackmann M., D'Auria F., Frassoni F., Neri A., Del Vecchio L., Musto P., Cilloni D, & Caivano A. (2017). EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells. Cellular oncology (Dordrecht, Netherlands), 40(5).

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