HUVECs were trypsinised and resuspended in ECM culture medium containing 0,6 gr/L methylcellulose (Sigma Aldrich). Cells were seeded (400 cells per well in 100 µl) in U-bottom 96 well plates (Costar) and cultured for 24 hours at 37°C and 5 % CO 2 to allow for formation of spheroids. The spheroids were collected and resuspended in FBS (Sciencell) containing 2,4 gr/L methylcellulose and mixed 1:1 with collagen I solution containing 3,77 g/L collagen I (Corning, USA), 10 % M199 medium (Sigma Aldrich), 0,018 M HEPES and 0.2 M NaOH to adjust pH to 7,4. The mixture with the spheroids was allowed to polymerize for 30 minutes in a 24 well plate. To induce sprouting, 100 µl of ECM with or without VEGF (50 ng/mL, Preprotech) was added to the gels. Spheroids were xed after 24 hours using 10 % fomaldehyde in PBS and visualized using an Olympus IX50 microscope. The cumulative sprout length per spheroid was measured using ImageJ software.
Collagen 1
Manufactured by Corning
Sourced in United States, Germany, United Kingdom, France
Corning Collagen I is a purified extracellular matrix protein derived from bovine sources. It is a key structural component of connective tissues and supports cell attachment, migration, and differentiation in various cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (15 mentions)
Fibronectin, by Corning (7 mentions)
Methylcellulose, by Merck Group (7 mentions)
Fibronectin, by Merck Group (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Laminin, by Corning (6 mentions)
M199 medium, by Merck Group (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Matrigel, by BD (5 mentions)
Collagen 4, by Corning (4 mentions)
Laminin, by Merck Group (4 mentions)
Methylcellulose, by Corning (4 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by ScienCell (4 mentions)
Bz x710 microscope, by Keyence (4 mentions)
Fibronectin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Axiovert microscope, by Zeiss (4 mentions)
Mito serum extender, by Corning (3 mentions)
141 protocols using collagen 1
1
Spheroid-based Angiogenesis Assay
2
Endothelial Cell Spheroid Angiogenesis Assay
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HUVECs were trypsinised and resuspended in ECM culture medium containing 0,6 gr/L methylcellulose (Sigma Aldrich). Cells were seeded (400 cells per well in 100 µl) in U-bottom 96 well plates (Costar) and cultured for 24 h at 37 °C and 5% CO2 to allow for formation of spheroids. The spheroids were collected and resuspended in FBS (Sciencell) containing 2,4 gr/L methylcellulose and mixed 1:1 with collagen I solution containing 3,77 g/L collagen I (Corning, USA), 10% M199 medium (Sigma Aldrich), 0,018 M HEPES and 0.2 M NaOH to adjust pH to 7,4. The mixture with the spheroids was allowed to polymerize for 30 min in a 24 well plate. To induce sprouting, 100 µl of ECM with or without VEGF (50 ng/mL, Preprotech) was added to the gels. Spheroids were fixed after 24 h using 10% fomaldehyde in PBS and visualized using an Olympus IX50 microscope. The cumulative sprout length per spheroid was measured using ImageJ software.
3
Collagen-Laminin Hydrogel Fabrication
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Unless otherwise stated, gels were fabricated from collagen I (Corning) ranging from 4 to 7 mg mL−1 neutralized with sodium hydroxide. laminin/entactin (Corning) was added to collagen I gels at densities of 0.25–1 mg mL−1. collagen I gels were formed at 37 °C for 30 min and then coated with 50 µg mL−1 of proteins and heparan sulfate proteoglycans at 37 °C overnight. The thickness of the gels was 300–500 µm. Where indicated, the gels were cross-linked with 20 mM genipin (Sigma) for 2 h and washed with PBS for 24–48 h at room temperature before coating. genipin is a low molecular weight cross-linker that has been used previously to enhance vascular stability and increases the stiffness of collagen fivefold [29 (link)]. collagen I gels were coated with collagen IV (Sigma), fibronectin (Life Technologies), laminin (Sigma), agrin (R&D Systems), and/or perlecan (R&D Systems).
4
HUVEC Spheroid-Sprouting Angiogenesis Assay
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Human umbilical vein endothelial cells (HUVECs) were trypsinized and resuspended in ECM culture medium (Sciencell, USA) containing 0.6 gr/L methylcellulose (Sigma). Cells were seeded (400 cells per well in 100 µl) in U-bottom 96 well plates and cultured for 24 h at 37 °C and 5% CO2 to allow for the formation of spheroids. The spheroids were collected and resuspended in FBS (Sciencell) containing 2.4 gr/L methylcellulose and mixed 1:1 with collagen I solution containing 3.77 g/L collagen I (Corning, USA), 10% M199 medium (Sigma), 0.018 M HEPES, and 0.2 M NaOH to adjust pH to 7.4. The mixture with the spheroids was allowed to polymerize for 30 min in a 24 well plate. A total of 100 µl of conditioned medium with or without VEGF (50 ng/mL) (Preprotech) was added to the gels. Spheroids were visualized after 24 h using an Olympus IX50 microscope. The cumulative sprout length per spheroid was measured using ImageJ software.
5
Adhesion Assay for Bone Marrow Stromal Cells
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Forward primer (5'-3') Reverse primer (5'-3')
of bands were determined using Image J (version 1.52a). Band intensities of α-SMA and FAK were normalised to β-actin and p-FAK was normalised to FAK. Percentages of MC1 and control intensities were compared by paired Wilcoxon test.
Assay 10: adhesion assay BMSC adhesion to fibronectin-coated, collagen I-coated and uncoated surface was assessed. 96-well plates were coated overnight at 4 °C using 16 µg/ mL fibronectin or 0.75 mg/mL collagen I (Corning).
fibronectin-and collagen I-coated wells were blocked for non-specific binding with 1 % heat-inactivated BSA for 1 h at 37 °C. 2,500 cells/well were seeded in sextuplets. After 15 min, 30 min and 4 h, cell suspension was removed and non-adherent cells were washed away with PBS. Adherent cells were fixed using 4 % neutral buffered formalin and stained with Hoechst 33342 (ThermoFisher Scientific). 4 images per well were taken at predefined spots using a Nikon Eclipse Ti2 upright brightfield microscope. Cells were counted manually using ImageJ. Cell counts at 15 min and 30 min were normalised to the respective 4 h count. Percentage increase of adherent MC1 and control BMSCs between 15 min (settling time) and 30 min were calculated and compared using a paired t-test.
of bands were determined using Image J (version 1.52a). Band intensities of α-SMA and FAK were normalised to β-actin and p-FAK was normalised to FAK. Percentages of MC1 and control intensities were compared by paired Wilcoxon test.
Assay 10: adhesion assay BMSC adhesion to fibronectin-coated, collagen I-coated and uncoated surface was assessed. 96-well plates were coated overnight at 4 °C using 16 µg/ mL fibronectin or 0.75 mg/mL collagen I (Corning).
fibronectin-and collagen I-coated wells were blocked for non-specific binding with 1 % heat-inactivated BSA for 1 h at 37 °C. 2,500 cells/well were seeded in sextuplets. After 15 min, 30 min and 4 h, cell suspension was removed and non-adherent cells were washed away with PBS. Adherent cells were fixed using 4 % neutral buffered formalin and stained with Hoechst 33342 (ThermoFisher Scientific). 4 images per well were taken at predefined spots using a Nikon Eclipse Ti2 upright brightfield microscope. Cells were counted manually using ImageJ. Cell counts at 15 min and 30 min were normalised to the respective 4 h count. Percentage increase of adherent MC1 and control BMSCs between 15 min (settling time) and 30 min were calculated and compared using a paired t-test.
6
HUVEC Spheroid Sprouting Angiogenesis Assay
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After 24-h transfection, HUVECs were trypsinized and resuspended in ECM culture medium containing 0.6 g/L methylcellulose (Sigma Aldrich). Spheroids were formed by seeding HUVECs (400 cells per well in 100 μL) in U-bottom 96-well plates (Costar) and culturing for 24 h at 37 °C and 5% CO2. Spheroids were resuspended in FBS (Sciencell) containing 2.4 g/L methylcellulose (dissolved in ECM) and mixed 1:1 with collagen-I solution (3.77 g/L collagen-I (Corning, USA), 10% M199 medium (Sigma Aldrich), 0.018 M HEPES, and 0.2 M NaOH to adjust pH to 7.4). In a 24-well plate, 1 mL of the mixture was added per well and polymerized for 30 min at 37 °C. In total, 100 μL of ECM with or without VEGF (50 ng/mL, Preprotech) was added. Spheroids were fixed after 24 h using 10% formaldehyde in PBS and visualized using a Zeiss DMIL LED microscope. The cumulative sprout length per spheroid was measured using ImageJ software.
7
Dual-Crosslinked Thiol-Hyaluronic Acid Hydrogels
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THA with a degree of substitution of 6% was synthesized as described previously [45 (link)]. THA was reconstituted (25 mg/ml) and photoinitiator Eosin Y (0.02 mg/ml, Sigma Aldrich) was added. THA (25 mg/ml final concentration) and THA-collagen (THA 12.5 mg/ml and 2.5 mg/ml collagen 1 isolated from rat tails, Corning, Bedford, USA) hydrogels were enzymatically crosslinked using peroxidase from horseradish (0.3 U/ml, Sigma Aldrich) and hydrogen peroxide (120 ppm, Carl Roth, Karlsruhe, Germany) with subsequent photo-crosslinking (10 min, 3 cm distance, AVIDE lamp, Well-Com Vertriebs GmbH, Gelsenkirchen, Germany) [34 (link)]. The variable for the two THA formulations was the addition of the fibrillary component (THA-col) compared to THA alone.
8
Quantifying Cell Migration Using Scratch Assay
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To analyze cell migration, the scratch wound migration assay and IncuCyte Zoom live cell imaging system (Essen Bioscience, Michigan, USA) were used. Cells were seeded into 96-well ImageLock plates (Essen Bioscience) and grown to confluence under standard conditions. As Caco-2 cells only loosely adhered to the well surface, ImageLock plates were pretreated with 5 μg collagen-1 (Corning, NY, USA) per well for 1 h at room temperature and washed with PBS before seeding. After 24 h, scratches were created simultaneously in all wells with a WoundMaker (Essen Bioscience) according to the manufacturer’s instructions. Wound closure was scanned every 4 h and monitored over 16 h using the Wound Width Analysis IncuCyte Software, allowing the exact identification of wound regions. The distance of cell migration in μm was calculated by subtracting the detected wound width from the initial wound width and dividing by 2. The experiment was performed in triplicate.
9
Cell Adhesion and Viability Assay
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Experiments were performed as previously reported (13 (link)). Briefly, 4×104 cells were plated in 100 µl of serum-free RPMI 1640 in 96-well microtiter plates coated with fibronectin, laminin, collagen 1, or collagen 4 (Corning Inc.). Then the culture plates were centrifuged at 500 rpm for 30 sec and incubated at 37°C in a humidified atmosphere with 5% CO2 for 2 h. After incubation, the plates were washed three times with assay buffer to remove non-adherent cells. Then, the adherent cells were evaluated for cell viability using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). Absorbance was measured at 490 nm using a microplate reader, Viento 808 IU (BioTek Instruments, Inc., Winooski, VT, USA). Three individual experiments were performed in triplicate.
10
Optimized Islet Encapsulation for Diabetes Therapy
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Human islets were received through the Integrated Islet Distribution Program (IIDP) and experiments were initiated within 24 h of receipt. On day 0, islets were counted and plated in suspension or in hydrogel co-culture. Islets were combined with hP-HG (8 mg/mL) at a density of 100 IEQ/10μL of hydrogel. The mixture was pipetted into 5 μL droplets in the bottom of an untreated petri dish, inverted, and incubated at 37 °C and 5% CO2 for 30 min. The polymerized droplets were moved into 24-well ULA plates (Corning, Corning, NY) for culture for 1–7 days in PIM(R) medium (Prodo Labs, Aliso Viejo, CA), at which point they were collected for MTS, GSIS, or total insulin content. In parallel, islets were cultured in suspension in 24-well plates for the same period of time. Islets embedded in collagen 1 (8 mg/mL) (Corning, Corning, NY) followed the same protocol as hP-HG. Islets were embedded in alginate following methodology adapted from Alagpulinsa et al.32 (link). Briefly, islets were mixed with 1.6% w/v sodium alginate in 150 mM NaCl, at 100 IEQ/10 μL, and manually dropped into a 100 mM CaCl2 bath 5 μL at a time to form droplets.
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