The NS5A transfection was repeated today. The experiment will still run for 48 hours and will still be monitored every 12 hours. Instead of splitting the cells to stimulate, twice as many cells were transfected. This experiment also transfected a batch of cells that will be used for flow cytometry, 18 million cells were used in this experiment, 3 mock transfections and 3 NS5A transfections.
To prepare the cells for flow cytometry:
Flow samples were taken at T=0 (after transfection the cells had about an hour to recover) and T=48
1.5 mL of cells were harvested, centrifuged (1000 rpm, 3 min) and the cell pellet was vortexed with .5mL of cold PBS. Another .5mL of cold PBS was added, the pellet was completely resuspended and the cells were centrifuged again. The cell pellet was vortexed with .2mL of cold PBS and added dropwise to .750mL of cold 75% ethanol. The cells incubated on ice for 30 minutes before being transferred to the fridge. Cells can be stored in this fashion for up to 1 week.