The WEHI 231 cells were transfected with NS5A-mCherry. 12 hours after stimulation cells were stimulated with anti-IgM. At T=36 cells were collected and stained with either ER tracker or Bodipy. Cells were then prepared for flow cytometry
Cells from experiments that are to be analyzed via Flow Cytometry or Electron Microscopy are prepared in the following manner:
Harvest cells and centrifuge (1000 rpm, 3 min), vortex the cell pellet with .5mL of cold PBS. Add another .5mL of cold PBS and completely resuspend the pellet. Centrifuge the cells again. Vortex the cell pellet was with .2mL of cold PBS and add the cells dropwise to .750mL of cold 75% ethanol. Incubate the cells on ice for 30 minutes before being transferring to the fridge. Cells can be stored in this fashion for up to 1 week.
Harvest 1.5 mL of cells and centrifuge off media (1000 rpm, 5 min). Wash twice with 500 uL of PBS. Fix cells with 150 uL of either 2% paraformaldehyde or cold 75% ethanol for 10 minutes in the fridge. Resuspend cell pellet in PBS.
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.