Transfected HUVECs were detached from the cell culture plates using trypsin–EDTA solution, and subsequently suspended in ECM medium enriched with 10% FBS. Following this, a total of 8 × 103 cells were cautiously mixed with 4 ml of cell culture medium and 1 ml of methocel stock solution, which contained 12 mg of methylcellulose (Aladdin, C104984-250 g). This resulted in the formation of spheroids, which were obtained by placing 25 μl drops of the cell solution onto the lid of a cell culture dish (100 × 20 mm), and then incubating the dish upside-down in a humidified cell culture incubator for 24 h. The hanging drops were then carefully washed off using phosphate-buffered saline. Subsequently, the HUVECs were resuspended in 2 ml of methocel solution containing FBS, and 1 ml each of PBS and rat collagen I (R&D system, Shanghai, China) was added gently. The resulting spheroid-collagen solution was added to 24-well plates (1 ml per well) for 24 h. The sprouts were captured using an inverted microscope (Olympus Corp, IX73P1F) and the number and average length of each sprout per cytosphere were determined using the Sprout Morphology plugin in Fiji (ImageJ).
Rat collagen 1
Manufactured by R&D Systems
Sourced in China
Rat collagen I is a type of extracellular matrix protein derived from rat tissue. It is a key structural component of various connective tissues in the body.
Automatically generated - may contain errors
Lab products found in correlation
Ix73p1f, by Olympus (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Axio observer a1 microscope, by Zeiss (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Dutscher (1 mentions)
Primocin ant pm 1, by InvivoGen (1 mentions)
Triton x 100, by Corning (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
7 protocols using rat collagen 1
1
HUVEC Spheroid-Collagen Sprouting Assay
2
Quantifying Lung Cell Migration in Collagen
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MACS-sorted PDGFRα+ cells from P6 lungs were counted and embedded in rat collagen I (R&D, 1 mg/mL) at 10,0000/well in 24-well plates. The collagen gels were incubated at 37°C for 20 minutes and transferred into 12-well plates with 1 mL DMEM/10% FBS (Gibco) medium. The pictures of gels were taken using ChemiDoc imaging system (Bio-Rad) every 24 hours. Areas of collagen gels from day 5 of culture were measured using FIJI software and compared with those from day 0.
3
HUVEC Spheroid Formation and Sprouting Assay
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HUVECs were detached from cell culture plates by trypsin-EDTA solution and were resuspended in compete medium (with 10% FBS). Thereafter, 4 ml of cell culture medium containing 8 × 103 cells was mixed carefully with 1 ml of methocel stock solution, containing 12 mg of methylcellulose (Sigma-Aldrich). Next, 25 μl of drops of the cell solution was placed onto the lid of a cell culture dish (100 × 20 mm) and incubated upside-down in a humidified cell culture incubator for 24 hours, and the spheroid shape was formed. The hanging drops were then washed off by phosphate-buffered saline, and HUVECs were resuspended with 2 ml of methocel solution containing FBS. Afterward, 2 ml of rat collagen I (R&D system, Shanghai, China) was added softly. The spheroid-collagen solution was added to 12-well plates (1 ml per well) for 24 hours. The sprouting assays were stopped by adding 1 ml of 10% paraformaldehyde. Sprouts were photographed under a Zeiss Axio Observer A1 microscope. The sprout number and the length of each sprout were recorded.
4
Monocyte-Endothelial Cell Interaction Protocol
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Purified human primary monocytes were cultured in RPMI 1640 (Gibco) supplemented with 2% FBS (Dutscher) and 1% Penicillin–Streptomycin (Gibco). Cells were cultured into round-bottom tissue culture-treated 96-well plates. The human Cerebellar Microvascular Endothelial Cells D3 (hCMEC/D3) cells34 (link) (provided by S. Bourdoulous, Institut Cochin, France) were cultured by using EndoGRO‐MV Complete Culture Media (Millipore) supplemented with 1 ng/mL bFGF (Sigma) and 1% Penicillin–Streptomycin (Gibco). Cells were plated and cultured in dishes coated with rat collagen-I (R&D Systems). Lithium Chloride (10 mM) and Resveratrol (10 μM) were added when seeding the cells in the culture inserts (cf. transmigration assay). Vero cells (ATCC, CCL-81) were cultivated in High-glucose DMEM media with L-Glutamine (Hyclone), 10% FBS, and 1% Penicillin–Streptomycin. All cell types were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
5
Cell Culture and Tissue Engineering Protocol
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The following reagents were purchased from Sigma-Aldrich: propylene glycol monomethyl ether acetate (PGMEA, 484431), chlorotrimethylsilane (386529), poly-L-ornithine hydrobromide (P3655), 3-aminopropyl)triethoxysilane (APTES; 440140), SB202190 (S7067), Corning® Matrigel® growth factorreduced product (356231), acetylcysteine (NAC; 1009005), nicotinamide (N0636), gastrin I (05–23–2301) and Triton™ X-100 (X100). The following reagents were purchased from Thermo Fischer Scientific: bis-(sulfosuccinimidyl) suberate (Bs3; 21580), ethylenediaminetetraacetic acid, (EDTA; J15694.AE), Neurobasal A Medium (10888022), GlutaMAX (35050061), Advanced Dulbecco’s Modified Eagle Medium (ADMEM; 12491015), N2 supplement 100X (17502048), B27 supplement 50X (A1486701), Antibiotic/Antimycotic 100X (MT30004CI), and TrypLE™ express enzyme 1X (12604021). Rat collagen I, lower viscosity (3 mg/mL, 3443100–01) and epidermal growth factor (EGF, 236-EG) were purchased from R&D Systems. TGF-β1 inhibitor (A83–01; 2939/10) was purchased from Tocris and Y-27632 (A3008) from APExBio. Primocin (ant-pm-1) was purchased from Invivogen. Polystyrene beads of 16.3 (SVP-150–4) and 101 μm (SVP-1000–4) were purchased from Spherotech Inc. Sylgard 184 poly(dimethylsiloxane) (PDMS) kit (2065622) was purchased from Ellsworth and all SU-8 resists (2002, 2025, 2050, 2100) from Kayaku Advanced Materials.
6
Seahorse Analysis of Mature Adipocytes
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Mature adipocytes differentiated from the supraclavicular BAT of one donor underwent Percoll density gradient centrifugation on day 10 of differentiation and were seeded on a seahorse 96-well plate (5,000 cells per well). For adipogenic cells, the wells were pre-coated with rat collagen I (3447-020-01, R&D systems) for 1 h at 37 °C in a cell culture incubator to support adherence. Seahorse extracellular flux measurements were performed 24 h after seeding as described below.
7
Collagen-Coated Cell Culture Protocol
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In order to cultivate cells on collagen-coated cell culture filter inserts, 5 µg cm 2 -1 of rat collagen I, lower viscosity, (3 mg ml -1 stock; R&D Systems, Minneapolis, MN, USA) was used to coat the filter membrane. The coated filters were incubated for 1 h in a humidified incubator at 37 °C and 5% CO 2 . Subsequently, the filters were rinsed twice with DPBS (Gibco by Life Technologies, Carlsbad, USA) before the PCP-R cell suspension was applied. Seeding and cultivation of the cells was performed as described for the "conventional" protocol.
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