Fixing Cells for Microscope/Flow Cytometry Work

Goal

• Goal: To fix cells (kill and preserve cell structures) so that they can be viewed using a microscope or a flow cytometer.

Pre-Protocol questions

First, answer these questions:

• Log phase growth is best for viewing cells; are your cells actively growing?
• If these cells have a fluorescent protein in their genome, are they actively making it? Do they need to be in log growth phase for expression? Do they need to be induced by another chemical, such as IPTG?

Protocol

Protocol (Version 1 / Jan 2023):

1. If your cells are not in liquid media, put them into sterile 1x PBS. Vortex to get cells into solution.
2. Spin down cells (250ul should be enough) using the microcentrifuge at full speed for 1 minute. The hinge of the microcentrofuge tube should be facing out, as you might not be able to see a pellet after spinning (and that's OK)
3. Pipet out all media from tubes. Place the pipette tip opposite of where the cell pellet should be.
4. Add 100ul of 4% paraformaldehyde (found on chemical shelves).
5. Set a timer for 15 minutes. Immediately vortex cells for 1 minute at high speed.
6. At the end of the 15 minutes, spin down cells using the microcentrifuge at full speed for 1 minute.
7. Pipet out the paraformaldehyde AND place the used paraformaldehyde into a microcentrifuge tube. Used paraformaldehyde from multiple samples can be pooled. It needs to be disposed in a specific chemical waste location (ask Eric about this!).
8. Add 250ul of sterile 1x PBS. Vortex cells for 1 minute. The time between first adding paraformaldehyde and this step cannot be longer than 20 minutes (inclusive of the 15 minute incubation)
9. These cells are now dead / fixed; you can remove them from the lab and take them to the microscopes / flow cytometer outside of the MEE lab.