A 3D spheroid-based angiogenesis assay was performed according to the procedure described by Heiss35 (link) with a slight modification. After one passage, HUVECs were plated for siRNA or DNA transfection. Then, 24 h after transfection, cells were suspended in sterile 0.24% w/v methylcellulose solution in EGM2 medium with 10% FBS and aggregated overnight in hanging drops (25 μL) to form cellular spheroids (500 cells/spheroid). Spheroids were embedded into 1 mg/mL bovine acid-treated collagen gels (Nippi, Tokyo, Japan). Vessel sprouting was stimulated with 20 ng/mL (final concentration) VEGF165 (Sigma-Aldrich, MO, USA) 30 min after embedding the spheroids. After a 24 h incubation at 37 °C, spheroids in collagen gels were fixed with 10% paraformaldehyde and in vitro angiogenesis was quantified using cellSens Standard2 software (Olympus, Japan) to measure the cumulative sprout length (CSL) of capillaries, defined by the presence of lumens, that grew from the spheroids after 3 separate HUVEC transfections. When HUVECs or HDMECs were transfected with BOSβgal, whole-mount X-gal staining of spheroids was performed according to the protocol described in5 (link).
Bovine acid treated collagen gels
Manufactured by Nippi
Sourced in Japan
Bovine acid-treated collagen gels are a type of lab equipment used for various applications. They are derived from bovine (cow) collagen that has been treated with acid, resulting in a gel-like substance. These gels can be used as a substrate or scaffold for cell culture, tissue engineering, and other research purposes.
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Lab products found in correlation
2 protocols using bovine acid treated collagen gels
1
Spheroid-Based Angiogenesis Assay
2
3D Spheroid-Based Angiogenesis Assay
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A 3D spheroid-based angiogenesis assay was performed according to the procedure described by Heiss 35 (link) with a slight modification. After one passage, HUVECs were plated for siRNA or DNA transfection. Then, 24 hours after transfection, cells were suspended in sterile 0.24% w/v methylcellulose solution in EGM2 medium with 10% FBS and aggregated overnight in hanging drops (25 μL) to form cellular spheroids (500 cells/spheroid).
Spheroids were embedded into 1 mg/mL bovine acid-treated collagen gels (Nippi, Tokyo, Japan). Vessel sprouting was stimulated with 20 ng/mL (final concentration) VEGF165 (Sigma-Aldrich, MO, USA) 30 min after embedding the spheroids. After a 24 hr incubation at 37 °C, spheroids in collagen gels were fixed with 10% paraformaldehyde and in vitro angiogenesis was quantified using cellSens Standard2 software (Olympus, Japan) to measure the cumulative sprout length (CSL) of capillaries, defined by the presence of lumens, that grew from the spheroids. In case of HUVEC transfection with BOSβgal, whole-mount X-gal staining of spheroids was performed according to the protocol described in 5 .
Spheroids were embedded into 1 mg/mL bovine acid-treated collagen gels (Nippi, Tokyo, Japan). Vessel sprouting was stimulated with 20 ng/mL (final concentration) VEGF165 (Sigma-Aldrich, MO, USA) 30 min after embedding the spheroids. After a 24 hr incubation at 37 °C, spheroids in collagen gels were fixed with 10% paraformaldehyde and in vitro angiogenesis was quantified using cellSens Standard2 software (Olympus, Japan) to measure the cumulative sprout length (CSL) of capillaries, defined by the presence of lumens, that grew from the spheroids. In case of HUVEC transfection with BOSβgal, whole-mount X-gal staining of spheroids was performed according to the protocol described in 5 .
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