All animal studies were approved and performed in accordance with guidelines established by Baylor College of Medicine Institutional Animal Care and Use Committees (IACUC). Severe combined immunodeficient mice (Jackson Lab, Bar Harbor, ME) were implanted in the cerebellum with 1 × 106 Daoy cells (ATCC, Manassas, VA) suspended in matrigel (Sigma-Aldrich, St. Louis, MO) to establish the medulloblastoma tumor model (n = 10). Glioma models were established in the caudate putamen. The human glioma model was established in Severe combined immunodeficient mice with 1 × 106 U87 (ATCC, Manassas, VA) cells suspended in matrigel (n = 8). The mouse glioma model was established in C57BL/6 Albino with 5 × 105 GL261 (ATCC, Manassas, VA) cells in matrigel (n = 17). Animals were monitored for general health and euthanized according to the established protocol.
Matrigel
Manufactured by American Type Culture Collection
Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is composed of extracellular matrix proteins, growth factors, and other components. Matrigel provides a reconstituted basement membrane that can be used as a substrate for culturing cells or for studying cellular processes.
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Lab products found in correlation
Matrigel, by Corning (4 mentions)
Matrigel, by BD (4 mentions)
Huvecs, by American Type Culture Collection (2 mentions)
Matrigel, by Merck Group (2 mentions)
Huvecs, by Olympus (1 mentions)
Inverted phase contrast microscope, by Leica camera (1 mentions)
Hpaec, by American Type Culture Collection (1 mentions)
Nu nu, by Jackson ImmunoResearch (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Severe combined immunodeficient mice, by Jackson ImmunoResearch (1 mentions)
Severe combined immunodeficient mice, by American Type Culture Collection (1 mentions)
Daoy cells, by American Type Culture Collection (1 mentions)
Gl261, by American Type Culture Collection (1 mentions)
Matrigel, by Olympus (1 mentions)
Wst 1 assay, by Merck Group (1 mentions)
Matrigel, by Lonza (1 mentions)
Ebm 2 medium, by Lonza (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Human egf, by Thermo Fisher Scientific (1 mentions)
11 protocols using matrigel
1
Preclinical brain tumor models
2
Colon Organoid Isolation and Expansion
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Isolation and expansion of mouse colon organoid cells was performed as described previously (Shiokawa et al., 2017) . Non-tumor epithelial cells obtained from male wild-type mice (Wt), male Apc Min/+ mice (Min), and tumor epithelial cells from male Apc Min/+ mice that were administrated with 2% DSS (w/v) (Tumor) were plated on a 24-well plate containing 50 ml of Matrigel (Corning) plus 200 ml E medium (DMEM/F12-GlutaMAX (GIBCO) supplemented with penicillin-streptomycin (GIBCO), 10 mM HEPES (GIBCO), 1x N-2 supplement (GIBCO), 1x B-27 supplement (GIBCO), 1x ITS-G (GIBCO), 1 mM N-acetylcysteine (Sigma) , 50 ng/mL human EGF (Invitrogen) and 1% bovine serum albumin (Wako)) supplemented with 10 mM Y27632 (Wako). The cells attached to the Matrigel were overlaid with 50 ml of Matrigel plus 500 ml of E medium supplemented with 10 mM Y27632 and a 1/3 volume of conditioned E medium prepared from L-WRN cells (ATCC, CRL-3276). The organoids were then maintained in Matrigel in E Medium supplemented with the conditioned medium.
3
BM-MPC Tubule Formation in Matrigel
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Matrigel (BD Biosciences, San Diego, CA) was thawed and placed in four-well chamber slides at 37°C for 30 min to allow solidification. BM-MPCs (1 × 105) were plated alone in Matrigel and incubated at 37°C under 21% or 1% oxygen for 18 h. BM-MPCs (5 × 104) were also coplated with 5 × 104 PKH26-labeled murine bEND.3 endothelial cells (American Type Culture Collection, Manassas, VA) in Matrigel and incubated under the same conditions. Tubule formation was defined as a structure exhibiting a length four times its width. Tubule counts were determined in five random fields per well using an inverted Leica DMIL microscope at ×40 magnification.
4
In Vitro Angiogenesis Assay with HUVECs
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Tube formation assay was utilized for angiogenesis examination in vitro with Matrigel (BD Biosciences, Franklin, NJ, USA) following the manufacturer's protocol.
Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 104 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium. After 6 h, tube formation of HUVECs was observed and calculated with an Olympus microscope.
Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 104 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium. After 6 h, tube formation of HUVECs was observed and calculated with an Olympus microscope.
5
Cord-like Structure Formation by HPAECs
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The formation of cord-like structures by human pulmonary artery endothelial cells (HPAECs [ATCC, Manassas, VA]) was assessed in Matrigel-coated wells [8] (link). HPAECs (40,000 cells/well) were seeded into 48-well plates coated with Matrigel (BD Biosciences, Mississauga, ON) into groups of triplicates: (1) room air, (2) room air+GYY4137 (100 microM) (3) hyperoxia (95% O2), (4) hyperoxia+GYY4137 (100 microM) and incubated at 37°C for 8 h. GYY4137 (morpholin-4-ium-4-methoxyphenyl (morpholino) phosphinodithioate) is a recently described slow-releasing H2S donor (Cayman chemical, Ann Arbor, Michigan). Cord-like structures were observed using an inverted phase contrast microscope (Leica, Richmond Hill, ON, Canada) and quantified by measuring the number of intersections and the length of structures in random fields from each well using OpenLab (Quorum Technologies Inc, ON, Canada).
6
Afatinib Treatment in Gastric Cancer Xenograft
7
Quantifying Capillary-like Structures Formation
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To measure tube formation, 48-well plates were coated with Matrigel (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions. Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC and cultured in DMEM-1640 and endothelial cells (ECs) differentiated from the CSCs (CSC-ECs) in DMEM/F12 supplemented with 0.2% FBS were seeded on Matrigel-coated plates (1.5×104 cells/well) and incubated for 4 h at 37°C. Subsequently, capillary-like structures were observed and were quantified by calculating the number of junctions per field; at least 5 different viewing fields were analyzed. All images were obtained using an inverted microscope (Olympus IX73; Olympus, Tokyo, Japan).
8
Generation and Maintenance of hiPSCs
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The hiPSCs (SCVI273) used in this study were kindly donated by the Joseph Wu Lab (Stanford Medicine, Department of Medicine and Radiology, Stanford CVI Biobank). Briefly, peripheral blood mononuclear cells were collected from a healthy donor, and the hiPSCs were generated by using the nonintegrative Sendai virus system.
Cells were cultured and maintained in a feeder-free system—hESC-qualified Matrigel™ (Corning) and TeSR1™ E8™ (STEMCELL Technologies Inc., Cambridge, MA) under standard culture conditions (37°C at 5% CO2). Briefly, we coated 100 mm Petri dishes with Matrigel™ for at least 1 hour at 37°C and plated 1 × 105 cells in TeSR™ E8™ media supplemented with ROCK Inhibitor Y-27632 (10 μM, ATCC) for 24 hours. The ROCK inhibitor was used to increase the cell survival by preventing dissociation-induced apoptosis (anoikis). The media was changed every day, and the cells were passaged using the cell dissociation recombinant enzymatic solution TrypLE™ Express (Gibco). Viable cells were determined by staining with trypan blue and counting in a hemocytometer.
Cells were cultured and maintained in a feeder-free system—hESC-qualified Matrigel™ (Corning) and TeSR1™ E8™ (STEMCELL Technologies Inc., Cambridge, MA) under standard culture conditions (37°C at 5% CO2). Briefly, we coated 100 mm Petri dishes with Matrigel™ for at least 1 hour at 37°C and plated 1 × 105 cells in TeSR™ E8™ media supplemented with ROCK Inhibitor Y-27632 (10 μM, ATCC) for 24 hours. The ROCK inhibitor was used to increase the cell survival by preventing dissociation-induced apoptosis (anoikis). The media was changed every day, and the cells were passaged using the cell dissociation recombinant enzymatic solution TrypLE™ Express (Gibco). Viable cells were determined by staining with trypan blue and counting in a hemocytometer.
9
3D Spheroid Culture of Human Keratinocytes
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SPT6 knock-down and wild-type human epidermal keratinocytes were cultured under 3D spheroid conditions (Ghosh et al., 2020 (link)) for colon-derived organoids. Briefly, primary human epidermal keratinocytes derived from human neonatal foreskin were used for all cell culture studies. Cells were seeded in Matrigel (Corning, 354234) domes at 5E4 cells per well in a 24 well plate. To allow for complete polymerization of the Matrigel, the plate was inverted and incubated at 37°C for 10 minutes before proliferation media (50% conditioned media prepared from L-WRN cells (ATCC, CRL-3276(Miyoshi and Stappenbeck, 2013 (link))) containing Wnt3a, R-spondin, and noggin) or differentiation media (5% conditioned media) was added to each well. Cells were maintained at 37°C/5% CO2 humidified conditions and media was changed every 2-3 days until spheroids were fully formed, and crypt budding was visible.
10
Angiogenic Potential of Extracellular Vesicles
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Tube formation assay was used to evaluate the angiogenic potential of EVs by coculturing with HUVECs on Matrigel (Corning, Belford, USA). For Matrigel tube formation assay, HUVECs were serum starved by culturing in epithelial cell basal medium-2 (ATCC) with 2% FBS for 5 hours. The serum starved cells were plated at the density of 4 × 10 4 cells per well on Matrigel, which coated the wells of 96-well plates and were equilibrated with EBM-2 medium (Lonza) (containing EVs with indicated concentrations). As a control, HUVECs were cultured alone and with PBS. When cultured on Matrigel, endothelial cells aligned themselves into a network structure within 12 hours and the results were documented photographically using an inverted microscope (Olympus) at 10× magnification and then the length of tube formation, node numbers were calculated using ImageJ Software (National Institutes of Health, Bethesda, Maryland). Proliferation was measured using a WST-1 assay according to the manufacturer's instructions (Sigma-Aldrich).
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