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Matrigel substrate

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Matrigel substrate is a soluble basement membrane extract derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is a complex mixture of extracellular matrix proteins, including laminin, collagen IV, heparan sulfate proteoglycans, and other growth factors. Matrigel substrate provides a physiologically relevant microenvironment for the culture and differentiation of various cell types, including stem cells, epithelial cells, and neurons.

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Lab products found in correlation

Mtesr1 medium, by STEMCELL (4 mentions) Stempro ezpassage tool, by Thermo Fisher Scientific (3 mentions) Stempro medium, by Thermo Fisher Scientific (2 mentions) Pmxs c myc, by Addgene (2 mentions) Pmxs hoct3 4, by Addgene (2 mentions) Pmxs sox2, by Addgene (2 mentions) Polybrene, by Merck Group (2 mentions) Heparin, by Merck Group (1 mentions) Stemline, by Merck Group (1 mentions) Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions) Dispase, by Thermo Fisher Scientific (1 mentions) Application suite program, by Leica camera (1 mentions) Severe combined immunodeficient mice, by Jackson ImmunoResearch (1 mentions) Gm00498, by Coriell Institute for Medical Research (1 mentions) Stemmacs ips brew xf, by Miltenyi Biotec (1 mentions) H14 wa14, by WiCell (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Pmxs klf4, by Addgene (1 mentions) Mrc 5 fibroblasts, by American Type Culture Collection (1 mentions) Pmxs nanog, by Addgene (1 mentions)

11 protocols using matrigel substrate

1

Generation and Characterization of SMA iPSC Lines

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Two independent control iPSC lines (4.2 and 21.5) and two independent SMA iPSC lines (3.6 and 7.12) were used [19] (link), [20] (link), [38] (link). iPSCs were grown in feeder free conditions on Matrigel substrate (BD Biosciences) in Nutristem medium (Stemgent). Neural stem cells (EZ Spheres) were generated by lifting intact colonies following dispase treatment (1 mg/ml, Gibco) and placing them directly into a human neural progenitor growth medium (Stemline, Sigma) supplemented with 100 ng/ml basic fibroblast growth factor (FGF-2, Millipore), 100 ng/ml epidermal growth factor (EGF, Miltenyi Biotec), and 5 µg/ml heparin (Sigma) in ultra-low attachment flasks and were passaged weekly using a chopping technique as previously described [39] (link).
Schwab A.J, & Ebert A.D. (2014). Sensory Neurons Do Not Induce Motor Neuron Loss in a Human Stem Cell Model of Spinal Muscular Atrophy. PLoS ONE, 9(7), e103112.
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2

Transwell-Based Tumor Cell Invasion Assay

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The transwell membrane with Matrigel Substrate (BD, USA) was used to investigate the invasion of tumor cells. 2 × 105 cells were added into the upper chambers, and the low chambers were filled with the RPMI‐1640 supplemented with 10% FBS. The cells were calculated for 24 hours at 37°C in 5% CO2. The cells under the surface of the lower chamber were fixed and stained with 0.1% crystal violet. The number of cells was observed from 5 randomly selected photographs under a microscope (magnification, ×200). The arrays were performed in triplicate.
Zhang X., Zhang T., Li C., Zhang M, & Chen F. (2018). CD164 promotes tumor progression and predicts the poor prognosis of bladder cancer. Cancer Medicine, 7(8), 3763-3772.
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3

Induced Pluripotent Stem Cell Lines for Huntington's Disease Modeling

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Pluripotent stem cell lines from HD patients (iPSHD11, iPSHD22, iPSHD34) and healthy controls (iPSRG2L, endo-iPS12, hESM01) were cultured in mTeSR1 medium (Stemcell Technologies, Canada) on a Matrigel™ substrate (BD Biosciences, USA) as described by the manufacturer. Striatal neurons were differentiated and characterized as described previously[10 (link)]. Study was approved by local ethics committee.
Nekrasov E.D, & Kiselev S.L. (2018). Mitochondrial distribution violation and nuclear indentations in neurons differentiated from iPSCs of Huntington’s disease patients. Journal of Stem Cells & Regenerative Medicine, 14(2), 80-85.
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4

Angiogenesis Evaluation of CHI3L1

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A tube formation assay was performed to evaluate the effect of CHI3L1 on the angiogenesis ability of HUVECs. In brief, the cells were cultured in a medium containing 10% of FCS pre-cultured with MEK inhibitor, PI3K inhibitor for 1 h, and corresponding shRNAs for 48 h, followed with or without 400 ng/ml of CHI3L1 for 24 h. Finally, the cells seeded into a 96-well plate at a density of 5 × 103 cells/well were overlaid with Matrigel substrate (BD Biosciences, CA), and were incubated at 37°C for 6 h. Capillary tube structures were observed and quantified (Hou et al., 2017 (link)). Representative images were captured using Leica Application Suite Program (magnification ×100). The value of tubular branches representing the capacity of angiogenesis was determined using Image J software (National Institutes of Health, Bethesda, MD, USA).
Xue Q., Chen L., Yu J., Sun K., Ye L, & Zheng J. (2021). Downregulation of Interleukin-13 Receptor α2 Inhibits Angiogenic Formation Mediated by Chitinase 3-Like 1 in Late Atherosclerotic Lesions of apoE−/− Mice. Frontiers in Physiology, 12, 690109.
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5

Fibroblast to iPSC Reprogramming Protocol

Cited 1 time
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Fibroblasts were generated from skin biopsies collected by K.A.R. and L.A.W. in patients previously described (28 (link)) and expanded by culturing in Dulbecco’s modified Eagle’s medium + 10% fetal bovine serum for 2 to 3 weeks. Two control lines, 162D and 165D, were from healthy siblings of NF1 patients. Line 13 was generated from fibroblast line GM00498 (Coriell). Fibroblast lines were reprogrammed with nonintegrative plasmids as previously described (37 (link)). Clonal colonies displaying iPSC morphology were manually selected and subsequently cultured on a Matrigel substrate (BD Biosciences) with mTeSR1 medium (Stem Cell Technologies). To determine pluripotency, teratomas were generated via three subcutaneous injections of 100,000 dissociated iPSCs in mTeSR1/30% Matrigel into nonobese diabetic mice/severe combined immunodeficient mice (Jackson Laboratory). After 2 to 3 months, tumors were surgically removed and sections were hematoxylin and eosin–stained by the Gladstone Histology Core at UCSF. All experiments were conducted with lines between passages 10 and 30.
Krencik R., Hokanson K.C., Narayan A.R., Dvornik J., Rooney G.E., Rauen K.A., Weiss L.A., Rowitch D.H, & Ullian E.M. (2015). Dysregulation of astrocyte extracellular signaling in Costello syndrome. Science translational medicine, 7(286), 286ra66.
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6

hESC Culture and Characterization

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Six hES cell lines-H1 (WA01), H7 (WA07), H9 (WA09), and H14 (WA14) (WiCell, Madison, WI, USA) and CM7 and CM14 (established at Galat lab [23 (link)])-were used for this study. For the microarray analysis H7, H9, H14, CM7, and CM14 cells were grown in StemPro medium (Invitrogen, Carlsbad, CA, USA) on a Matrigel substrate (BD Bioscience, San Jose, CA, USA). The confluent cultures of hESCs growing on 10-cm dishes were split to 6 experimental 10-cm dishes mechanically by using the StemPro EZ Passage tool (Invitrogen). For amino acid assays, H9 cells were maintained in DMEM/F12 and supplemented with Knockout Serum Replacement (SR-1), FGF, 2,β-mercaptoethanol, and GlutaMax, Gibco Inc., Billings, MT, USA). For RNAseq analysis, H1 cells were maintained on Matrigel-coated culture dishes in StemMACS iPS-Brew XF (Miltenyi Biotec, Bergisch Gladbach, Germany). A single-cell suspension was counted with the help of an automatic cell counter, Nexcelom (SelectScience, Church Farm Business Park, UK), during cell lifting for the analysis. Cell confluence and morphology were observed daily.
Van Winkle L.J., Galat V, & Iannaccone P.M. (2020). Lysine Deprivation during Maternal Consumption of Low-Protein Diets Could Adversely Affect Early Embryo Development and Health in Adulthood. International Journal of Environmental Research and Public Health, 17(15), 5462.
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7

Matrigel Invasion Assay Protocol

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Cells (1 × 105/well) were seeded into the upper chamber, and 2 mL medium with 10% FBS was added to the lower chamber. Membrane with Matrigel substrate (BD Biosciences, Franklin Lakes, NJ, USA) was used for filtering. Then after culturing for 24 hours, cells filtered through the membrane were stained with 0.5% crystal violet solution and calculated under the microscope. Three repeat arrays were carried out.
Tian D., Wu Z., Jiang L., Gao J., Wu C, & Hu H. (2018). Neural precursor cell expressed, developmentally downregulated 8 promotes tumor progression and predicts poor prognosis of patients with bladder cancer. Cancer Science, 110(1), 458-467.
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8

Generation of iPSC-SR2 Line from MRC-5 Fibroblasts

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The iPSC-SR2 line was derived from MRC-5 fibroblasts (ATCC) by overexpressing Oct4, Sox2, Nanog, and cMyc using retroviral vectors (pMXs-cMyc, pMXs-Klf4, pMXs-hOct3-4, and pMXs-Sox2; Addgene) [33 (link)]. The retroviral vectors were produced by transient transfection of 293 T cells. The fibroblasts were incubated in the viral supernatants containing 5 μg/ml polybrene (Sigma) for 4 h. The transduced cells were then incubated for 3 weeks until development of the pluripotent clones. After isolation the clones were grown in mTeSR1 medium (Stem Cell Technologies) on a Matrigel® substrate (BD Biosciences). The cultures were split mechanically using the StemPro EZ Passage tool (Invitrogen).
Galat Y., Dambaeva S., Elcheva I., Khanolkar A., Beaman K., Iannaccone P.M, & Galat V. (2017). Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential. Stem Cell Research & Therapy, 8, 67.
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9

Angiogenesis Assay with RCSEC Cells

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RCSEC cells (1 × 104 cells/well) were seeded onto a Matrigel substrate (356234, BD Biosciences, Corning) and incubated for 6 h. Lumen formation was observed under an optical microscope, and the tubular structure was analyzed and quantified using ImageJ software.
Lei W., Xu H., Yao H., Li L., Wang M., Zhou X, & Liu X. (2023). 5α-Hydroxycostic acid inhibits choroidal neovascularization in rats through a dual signalling pathway mediated by VEGF and angiopoietin 2. Molecular Medicine, 29, 151.
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10

Generation of iPSC Lines from Fibroblasts

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The iPSC-WT cell line was derived from MRC-5 fibroblasts (ATCC), and the iPSC-DS clones were derived from AG06872 fibroblasts (Coriell). The fibroblasts were transduced with retroviral vectors (pMXs-cMyc, pMXs-Nanog, pMXs-hOct3-4, and pMXs-Sox2; Addgene) to overexpress Oct4, Sox2, Nanog, and c-Myc transgenes. The retroviral vectors were produced by transient transfection of 293T cells. Following this, the fibroblasts were incubated for 4 h in the viral supernatants containing 5 μg/mL polybrene (Sigma). The transduced cells were then incubated for 3 weeks until development of the pluripotent clones. After isolation, the clones were grown in StemPro medium (Invitrogen) on a Matrigel® substrate (BD Biosciences). The cultures were split mechanically using the StemPro EZ Passage tool (Invitrogen).
Galat V., Galat Y., Perepitchka M., Jennings L.J., Iannaccone P.M, & Hendrix M.J. (2016). Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells. Stem Cells and Development, 25(14), 1060-1072.
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