To begin, arrange the required experimental materials on the working platform. Add 100 microliters of sterile 0.4%gelatin solution to the wells of a black walled 96 well clear bottom plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.
Under an inverted microscope, confirm that endothelial cells in a T75 flask are 80 to 100%confluent. Remove the DMEM complete medium from the flask via aspiration. To wash the cells, add 10 milliliters of PBS and gently shake the flask.
Replace the PBS with five milliliters of cell dissociation solution. Incubate the flask at 37 degrees Celsius and 5%carbon dioxide for approximately 12 minutes. Tap the flask after six minutes.
To help facilitate dissociation. Add five milliliters of the prewarmed DMEM complete medium to the flask, and mix the cell suspension. Using a 10 milliliter serological pipette, transfer the cell suspension into a sterile 50 milliliter tube and centrifuge at 150 G for five minutes.
In the meantime, using a pastor pipette connected to a vacuum, aspirate the gelatin solution from the 96 well plate. After centrifugation of the cells, remove the supernatin from the tube without disturbing the pellet. Using a serological pipette, add eight milliliters of fresh DMEM complete medium to the tube, and gently pipette the suspension to break up the cell pellet.
Count the cells using trippen blue on a hemocytometer. Add DMEM complete medium to make a density of 600, 000 cells per milliliter suspension. Mix the cell suspension by gently inverting the tube.
Add the cell suspension to a 50 milliliter multi-channel pipette reservoir. Every 30 seconds, gently rock the reservoir side to side to mix the suspension and use a multi-channel pipette to add 100 microliters to each well of the 96 well plate coated with gelatin. Replace the transparent plate cover and gently shake the plate by sliding it on the surface of the sterile hood from north to south and east to west.
Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 16 to 24 hours.
