To begin, prepare 10 times M199 culture medium by dissolving M199 powder to 10 times concentration in 90 milliliters of deionized water, followed by the addition of 10 milliliters of FBS. Filter the solution through a 0.22 micron filter. The filtered medium can be stored at four degrees Celsius for up to two months.
For preparing the matrix gel with vascular cells, use about 2 million vascular cells. Cool the sterile 1.5 milliliter tube containing the appropriately prepared cell pellet in an ice bath. Thaw the matrix gel completely in an ice bath without mixing or vortexing it.
Similarly, cool the prepared 10 times M199 culture media along with the test reagent or drug of interest to four degrees Celsius. Next, maintaining a volume ratio of 8.8 to 1 to 0.2, combine the pre-cooled matrix gel with the pre-cooled 10 times M199 media containing 10%FBS, and the pre-cooled test reagent to produce the matrix gel mixture. Finally, re-suspend the pre-cooled cells in 400 microliters of the prepared matrix gel mixture by mixing gently to avoid forming bubbles.
Keep the tube containing the cells in matrix gel on ice until the mice are ready for the matrix gel injection.
