Tube formation assays were performed using an in vitro angiogenesis kit (Gibco) according to manufacturer’s instructions. Briefly, the wells of a 48-well plate were coated with 100 μL of reduced growth factor Geltrex matrix (Gibco) and incubated at 37 °C for 30 min. Human umbilical vein endothelial cells (HUVECs) were diluted in spent media from unstressed or mechanically stressed RPE cultures to a concentration of 106 cells/mL. 200 μL of cell suspensions were seeded on Geltrex matrices and incubated for 6 h at 37 °C in a humidified incubator with 5% CO2 to induce endothelial tube formation. Next, HUVECs were stained with Calcein AM dye (Thermo Fisher Scientific) and imaged using an Eclipse TS100 fluorescence microscope (Nikon Instrument Inc., Melville, NY). Tube length and node numbers were quantified using the ridge detection plugin for ImageJ software [43 (link), 44 ].
In vitro angiogenesis kit
Manufactured by Thermo Fisher Scientific
The in vitro angiogenesis kit is a laboratory tool designed to study the formation of new blood vessels in a controlled, in vitro environment. The kit provides a standardized platform for researchers to investigate the cellular and molecular mechanisms involved in the angiogenesis process.
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2 protocols using in vitro angiogenesis kit
1
Endothelial Tube Formation Assay
2
Angiogenesis Tube Formation Assay
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Tube formation assay was performed using an in vitro angiogenesis kit (Gibco) according to manufacturer's instructions. Briefly, the bottom of the wells of a 48-well plate were coated with 100 µL of reduced growth factor Geltrex matrix (Gibco) and incubated at 37 ˚C for 30 minutes.
HUVECs were then diluted in spent media from control or mechanically stressed ARPE-19 cultures to make a concentration of 1.2510 5 cells/mL. 200 µL of cell suspensions were seeded on Geltrex matrices and incubated for 6 hours at 37 ˚C and in a humidified incubator with 5% CO2 to induce endothelial tube formation. Next, HUVECs were stained with Calcein AM dye (Thermo Fisher Scientific) and imaged using an Eclipse TS100 fluorescence microscope (Nikon Instrument Inc., Melville, NY). Tube length and node numbers were quantified using ImageJ software.
HUVECs were then diluted in spent media from control or mechanically stressed ARPE-19 cultures to make a concentration of 1.2510 5 cells/mL. 200 µL of cell suspensions were seeded on Geltrex matrices and incubated for 6 hours at 37 ˚C and in a humidified incubator with 5% CO2 to induce endothelial tube formation. Next, HUVECs were stained with Calcein AM dye (Thermo Fisher Scientific) and imaged using an Eclipse TS100 fluorescence microscope (Nikon Instrument Inc., Melville, NY). Tube length and node numbers were quantified using ImageJ software.
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