At approximately passage six, the neurospheres were plated onto GFR Matrigel (BD)-coated dishes in neurobasal medium (Life Technologies) containing 2 % B27 supplement (Life Technologies) and 2 mM L-glutamine (Life Technologies) (differentiation medium). For the PiggyBac vector transfection experiments, iPSC-derived neurospheres were used at stages beyond six passages when the regional identities could be caudalized and stably propagated. For differentiation in dissociated culture, the neurospheres were dissociated using TrypLE Select (Life technologies) for 5 min at 37 °C, and the dissociated iPSC-NPCs were seeded onto GFR-Matrigel (BD)-coated 96-well plates and 6-well plates in differentiation media.
Gfr matrigel
Manufactured by BD
Sourced in United States, Germany, United Kingdom
GFR) Matrigel is a complex extracellular matrix preparation isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is a gelatinous protein mixture secreted by these cells that resembles the complex extracellular environment found in many tissues.
Automatically generated - may contain errors
Lab products found in correlation
Versene solution, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mtesr1, by STEMCELL (5 mentions)
Chir99021, by Bio-Techne (5 mentions)
Accutase, by Merck Group (5 mentions)
Rock inhibitor y 27632, by Bio-Techne (4 mentions)
Blebbistatin b, by Merck Group (4 mentions)
Tryple, by Thermo Fisher Scientific (3 mentions)
μ angiogenesis slides, by Ibidi (3 mentions)
Dispase, by Thermo Fisher Scientific (3 mentions)
Collagenase xi, by Merck Group (3 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (3 mentions)
Rpmi b27 without insulin, by Bio-Techne (3 mentions)
Basic fibroblast growth factor (bfgf), by WiCell (3 mentions)
Mtesr1, by Merck Group (3 mentions)
Rpmi b27 without insulin, by Thermo Fisher Scientific (3 mentions)
Polybrene, by Merck Group (2 mentions)
Insulin transferrin selenium, by Corning (2 mentions)
24 well transwell insert with a 0.4 μm pore, by Corning (2 mentions)
Rock inhibitor y 27632, by Merck Group (2 mentions)
92 protocols using gfr matrigel
1
Differentiation of iPSC-Derived Neurospheres
2
3D Tumor Spheroid Nanoparticle Uptake
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3D tumor spheroids were prepared as described previously.60 (link) Briefly, cells were passaged onto 96-well round-bottom ultralow attachment plates (Corning). After 48–72 h, cell spheroids were transferred onto 18 mm glass coverslips coated with Matrigel GFR (BD) and cell media was supplanted with 50 μL·mL–1 of Matrigel GFR. Growth media was removed after 48 h and tumor spheroids were washed with PBS (Ca2+/Mg2+) of appropriate pH and incubated for 3 h in PBS containing 0.17 nM nM LbL nanoparticles. Following incubation, cells were rinsed with PBS, fixed in freshly prepared 3.7% formaldehyde (PBS, pH 7.4) for 15 min at RT, and permeablized with 0.1% Triton X-100 (PBS) for 5 min at RT. Coverslips were stained using PBS containing 0.66 μM Alexa Fluor 568 phalloidin (Life Technologies), 4 drops of NucBlue (DAPI; Life Technologies), and 1% BSA for 30 min at RT, then rinsed with PBS prior to mounting with Fluoromount (Sigma) onto no. 0 glass-bottom 35 mm MatTek dishes and imaging using a Nikon 1AR Ultra-Fast Spectral Scanning Confocal Microscope.
3
Differentiation Assay for PG Stem Cells
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To asses differentiation capacity of PG stem cells, differentiation assays were performed. At the end of a passage in self‐renewal, medium on top of gels was removed and the Matrigel was dissolved using cold 1× PBS to release organoids from the gel. The organoids were collected and pelleted by centrifugation. Pelleted organoids were resuspended into 10× DMEM, collagen I (Rat Tail, Corning, 354236), 1 M NaOH and Matrigel GFR (BD Biosciences) (40 collagen I:60 Matrigel GFR) mixing carefully. Next, the mix was plated immediately as 100 µl/well into a flat bottomed 96‐well plate (precoated with 40 μl of 50 DMEM/F12: 50 Matrigel) and allowed to solidify for 20 min at 37°C. After solidification, 150 μl of MM with 10% fetal calf serum (FCS), 1 μM DAPT (Sigma), and 50 ng/ml HGF (Peprotech/tebu‐bio) was added on top of each gel and incubated at 37°C. Medium was refreshed every 4th day. After 15 days in differentiation assay, the medium on top of the gels was removed, and the gels were collected into Eppendorfs containing 0.5 mg/ml Collagenase Type I and 0.5 mg/ml Dispase in DMEM/F12. Then, the Eppendorfs were placed in a shaking water bath at 37°C.
4
Gene Knockdown Using Retroviral and Lentiviral Vectors
5
Lung Organoid Formation from Lineage-Labeled Cells
6
HUVEC Tube Formation Assay
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HUVEC tube formation capacity was analyzed by using an Angiogenesis μ-slide system (IBIDI GmbH, Planegg/Martinsried, Germany). μ-slide wells were coated with 10 μl growth factor reduced (GFR) Matrigel (BD Biosciences) for at least 30 min at 37 °C. After matrigel polymerization, HUVECs at a density of 2 × 104 were plated and incubated at 37 ºC for 24 h in the presence of secretomes from MenSCs under N or AH conditions at 100 μg/ml concentration. Images were taken with an inverted microscope (Nikon Elipse TE2000-S) and analyzed by using Image J Software with an Angiogenesis Analyzer plug-in.
7
Murine Intestinal Organoid Culture
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Pieces of small intestine (~0.5 mm) from NOD-SCID IL2 receptor γ chainnull (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were incubated for 20 min at room temperature in Gentle Cell Dissociation Reagent (StemCells Inc., Newark, CA, USA). Isolated crypts were obtained through a 70-μm strainer and seeded into mixed solution with 1:1 growth factor-reduced (GFR) Matrigel (BD Biosciences) and IntestiCult™ Organoid Growth Medium (StemCells Inc.) in 24-well Clear TC-Treated Multiple Well Plates (Costar, Washington, DC, USA). The medium was changed every 3–4 days, and mIOs were passaged every 1 week. To evaluate the effects of murine IL-2 (mIL-2) on mIOs, organoids were treated with recombinant mIL-2 (1–20 ng/ml, Peprotech). Organoid morphology was observed using microscopy (DMI 4000B, Leica, Wetzlar, Germany).
8
Analyzing HUVEC Tube Formation on Matrigel
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Growth factor-reduced (GFR) Matrigel (BD Bioscience) was thawed on ice at 4 °C overnight. Ten microlitres of Matrigel was plated onto each inner well of the μ-angiogenesis slides (IBIDI, Germany) and allowed to solidify at 37 °C for 30 min. 1 × 104 HUVECs were reconstituted in CM only, CM containing anti-VEGF neutralizing antibody (10 μg/ml), CM with IgG isotype control (10 μg/ml), complete medium, EGM or SFM to a total volume of 50 μl and plated on the GFR Matrigel. The plates were incubated for 6 h in a humidified incubator at 5% CO2 and 37 °C. Images were taken using an inverted phase-contrast microscope (Nikon) under 4× and 10× objectives. The tube length was measured using WimTube (Wimasis, GmbH, Germany) from the 4× magnification images of three wells for each condition. The experimental samples and controls were assayed in triplicates.
9
Comprehensive Cell Migration Assays
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For migration assays, cells (6 × 104 cell/insert) were plated in the upper compartment of a Transwell tray (Corning, Acton, MA). Cell invasion assays were performed using BioCoat Matrigel Invasion Chambers (BD Bioscience, Franklin Lakes, NJ). For 3D invasion, cells were suspended in medium supplemented with 4% serum. Eight-chambered plates (BD Biosciences) were coated with Growth Factor Reduced (GFR) Matrigel™ (BD Bioscience). The cells (5 × 103 cells/ml) were mixed (1:1) with assay medium containing Matrigel (10%). For tracking, cells were seeded (3 × 103 cells/cm2) on collagen-coated μ-slide 8-well trays (Ibidi) in full medium without EGF. After overnight incubation, the medium was changed and time-lapse images were taken (3–5 h; 10-min intervals). The positions of cell nuclei were followed using ImageJ. For chemotaxis, cells (3 × 106 cells/ml) were seeded in the main chamber of a chemotaxis slide (Ibidi). As control, time-lapse images of cells were taken (6 h; 15-min intervals) in the absence of EGF. Thereafter, EGF was added to one of the side reservoirs, followed by another 6-h session of time-lapse imaging.
10
Teratoma Formation and Lineage Differentiation of iPSCs
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For in vivo differentiation of iPS Cells: 1×106 cells were resuspended in 50 μL 1:3 diluted GFR matrigel (BD Biosciences). The cell suspension was then injected into the left testis of a severe combined immunodeficiency (SCID) mouse, while the right testis was injected with 1:3 diluted GFR matrigel alone, as a negative control. Following teratoma formation, classic histological staining was carried out using Mayer’s Haematoxylin and Eosin. Images were taken using a Zeiss Axioscope Z Plus microscope.
For in vitro differentiation of iPS cells: iPS cells were grown for 10 days in DMEM supplemented with 20% FBS. Medium was changed once every three days, and cells were then fixed with 4% PFA. The following antibodies were used: anti-α-fetoprotein (endoderm), anti-βIII-tubulin (ectoderm), and anti-α-smooth muscle actin (mesoderm), all from Millipore. Images were taken using a Nikon Eclipse 50i microscope at x10 magnification.
For in vitro differentiation of iPS cells: iPS cells were grown for 10 days in DMEM supplemented with 20% FBS. Medium was changed once every three days, and cells were then fixed with 4% PFA. The following antibodies were used: anti-α-fetoprotein (endoderm), anti-βIII-tubulin (ectoderm), and anti-α-smooth muscle actin (mesoderm), all from Millipore. Images were taken using a Nikon Eclipse 50i microscope at x10 magnification.
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