Human induced pluripotent stem cell (iPSC)-derived GABAergic neurons and astrocytes (iCell® GABANeurons and Astrocytes) were purchased from FUJIFILM Cellular Dynamics, Inc. (FCDI, Madison, WI). Matrigel™ Growth Factor Reduced Membrane Matrix was purchased from Corning™ (Corning, NY). The Matrigel was diluted to 5 mg/mL with iCell Neural Complete Maintenance Medium (iCell Neural Base Medium 1 + 2% Neural Supplement A + 1% Penicillin/Streptomycin) on ice. Neurons and astrocytes were mixed in ratios of 4:1 and 1:4 (A1/N4 and A4/N1, respectively) and then embedded in Matrigel. 2 μL of cell-gel matrix was seeded into each gel lane of a 2-Lane OrganoPlate® (MIMETAS, Netherlands) using a repeater pipette (Eppendorf Repeater®, E3X, Hauppauge, NY) and then gelled at 37°C and 5% CO2 for 1 h. Next, 20 μL of medium was added to the gel lane, and 50 μL of medium was added to the inlet and outlet of the medium lane (100 μL total) of each well. The plate was incubated at 37°C and 5% CO2 on an interval rocker (MIMETAS, Netherlands) to allow medium perfusion with bi-directional flow. Medium was refreshed every other day.
Matrigel membrane matrix growth factor reduced
Manufactured by Corning
Sourced in United States
Matrigel™ Membrane Matrix Growth Factor Reduced is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. It is a gelatinous protein mixture that resembles the complex extracellular environment found in many tissues. This product is a reconstituted basement membrane without the addition of some growth factors.
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Lab products found in correlation
Bay 60 6583, by Bio-Techne (2 mentions)
Intesticult organoid growth medium, by STEMCELL (2 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (2 mentions)
Vegf165, by Thermo Fisher Scientific (1 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Caprylocaproyl macrogol 8 glycerides, by Gattefosse (1 mentions)
Polyoxyethylene 160 polyoxypropylene 30 glycol poloxamer 188 p188, by BASF (1 mentions)
Brij 35 solution, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Abcam (1 mentions)
Drabkin s reagent, by Merck Group (1 mentions)
5 protocols using matrigel membrane matrix growth factor reduced
1
Microtissue Modeling of iPSC-derived Neurons
2
3D Cyst Formation and Cell-in-Cell Dynamics
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MCF7 cells were seeded in 3D matrigel using established protocols (Durgan et al., 2011 (link)), and incubated to form early stage cysts (2–4 cells) in which matrix contacts are retained, to minimise the induction of detachment-induced entosis. Briefly, four-well, glass-bottomed chamber slides (Lab-Tek II; 155382) were coated with a thin layer of 80% Matrigel Growth Factor Reduced Membrane Matrix (Corning; 356230)/20% Rat Collagen I (Cultrex; 3440-100-01), then overlaid with 5 × 10∧4 cells in 2% Matrigel/media; 10 μM Y-27632 was included to suppress basal detachment-induced entosis during seeding. Cells were incubated for 24 hr to initiate cyst formation, Y-27632 was then washed out and replaced with fresh media −/+ RO-3306 (5 μM), to allow cell-in-cell formation to proceed in 3D, in the presence or absence of mitosis. Twenty-four hour later, cysts were formalin fixed, stained for actin (488-phalloidin) and DNA (Hoechst) and imaged by confocal microscopy.
3
Isolation and Culture of Murine Intestinal Crypts
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Proximal small intestines of VillinCre mice were flushed with ice-cold PBS. Tissue was incubated in 10mL of crypt isolation buffer (1xPBS-2mM EDTA) on a rocker for 5 min at 4°C. Tissue was cut longitudinally into small fragments roughly 0.8cm long and incubated in fresh isolation buffer on a rocker for 45 min at 4°C. After incubation, tubes were shaken vigorously until tissue floated, indicating removal of the mucosal layer. Cell suspensions were passed through 70μm cell strainer. Cells were washed with 1 × DMEM and then centrifuged at 50 × g for 10 min. Crypts were counted by hemocytometer and plated at 350 crypts per well in a 1:1 ratio of Matrigel™ Membrane Matrix Growth Factor Reduced (Corning, Cat #354230) to Advanced DMEM/F-12 (Thermo Scientific, catalog #12634-010). Enteroids were cultured for five days in IntestiCult™ Organoid Growth Medium (StemCell Technologies, Vancouver, Canada, catalog # 06005). Wells were treated with 10 μm of BAY 60–6583 (Tocris Bioscience, Bristol, UK, catalog# 4472) or vehicle control (DMSO) for 48 h. Organoids were harvested and lysed in RIPA buffer with 2× protease inhibitors and 2× EDTA (Thermo Scientific, catalog# 78444). Cell lysate protein concentration was determined using Bradford protein assay kit as explained above.
4
Angiogenesis Evaluation Protocol
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PMX disodium hemipentahydrate was purchased from Shipla Medicare (Karnataka, India). Caprylocaproyl macrogol-8-glycerides (Labrasol) were provided by Gattefossé (Saint Priest, France). Deoxycholic acid (DOCA), Drabkin’s reagent, and Duolink in situ mounting medium with DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). Polyoxyethylene (160) polyoxypropylene (30) glycol (poloxamer 188; P188) was obtained from BASF (Ludwigshafen, Germany). Matrigel membrane matrix (growth factor reduced) was purchased from Corning (Manassas, VA, USA). VEGF165 and basic fibroblast growth factor (bFGF) were purchased from Pepro Tech (Rocky Hill, NJ, USA). Brij-35 solution was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The solvents used in high-performance liquid chromatography (HPLC) were obtained from Merck and Thermo Fisher Scientific (Waltham, MA, USA). Polyethylene glycol-conjugated rat anti-mouse CD-31 antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-thrombospondin-1 antibody and Goat anti-rabbit Alexa Fluor 488 were purchased from Abcam (Cambridge, UK) and Invitrogen (Carlsbad, CA, USA), respectively.
5
Isolation and Culture of Murine Intestinal Crypts
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