Neoangiogenesis

Neoangiogenesis protocol

-         Cultivate mouse melanoma cell line B16F0 in complete Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientifc) supplemented with 5% (v/v) fetal bovine serum (FBS) and maintained at 37°C in a humidified incubator with 5% CO₂. 

-         Cell preparation: 100.000 B16F0 cells, or vehicle, with Trypan Blue (0.2%) in relation 1:1. Final volume: 50 μl.

-         Inoculate intradermally in the dorsal cephalad site of mice.

-         Seven days later, anesthetize each mouse with isoflorane inhalant and sacrifice them. 

-         Perform a longitudinal incision in the medial ventral line and open the skin of the inoculation site (take special care to not cut the inoculation site).

-         Put a 1.5 cm x 1.5 cm side squad centered on the injection site (identified as a blue point) and take pictures.

-         Count blood vessel bifurcations (each new branch) from the pictures using ImageJ software (National Institutes of Health, Maryland, USA, https://imagej.nih.gov/ij/).

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