Last week I discussed how to fix adherent cells for imaging. Fixing adherent cells was easy because you can directly grow then on your cover slips. However, suspension cells – which grow suspended in media and not on any surface – cannot be attached to your cover slips the same way. Read below to learn how to fix and ‘smear’ your suspension cells, using centrifugation, onto your cover slips for excellent imaging results.
Things You’ll Need:
|Supsension cell line||Pipettes & tips|
|Methanol, Ethanol or PFM||Cell incubator|
|Versene (optional)||Swing bucket centrifuge|
|Cover slips (see this article about cover slip sizes)|
Step #1: Use sterile techniques.
I said it before, and I am going to say it again: Use sterile techniques when doing cell culture. It is so much easier to prevent contamination than it is to clean it up once you have it. In short: spray everything that goes into the cell-culture hood with ethanol, only open your flasks in the hood, always wear gloves, and be careful not to cross contaminate by letting your pipette tips touch anything.
Step #2: Collect your cells.
Collect your suspension cell line as you normally would and centrifuge your cells down. Then aspirate off the old growth media.
Step #3: Re-suspend your cells.
Next you will need to re-suspend your suspension cells in PBS*, quantify, and dilute your cells to 2 x 106 cells/ml. Alternatively, if your suspension cells are prone to sticking together and forming unsightly clumps, you can re-suspend your cells in Versene Cell Dissociation Reagent instead of PBS*. Versene combined with some gentle pipetting should break up any cell clumps.
*PBS or phosphate buffered saline is a common buffer used in cell culture and histology. The osmolarity and ion concentration of PBS is the same as human tissues, so it will not cause your cells to shrivel or explode. PBS is 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, and 2 mmol KH2PO4 at pH7.4. Be sure to sterile filter your PBS.
Step #4: Fix your cells.
Add an equal volume of fixation solution to your cell/PBS or cell/Versene suspension. This fixation solution can be made of either organic solvents or paraformaldehyde (PFM) depending on your method of choice.
Organic solvent method: Organic solvents work to preserve your cells by removing lipids, dehydrating your cells, and denaturing and precipitating the proteins in your cells. If you choose this method, add equal amount of ice-cold methanol, ethanol or a 1:1 mix of ethanol and methanol to your 2 x 106 cell/ml suspension to create a 1 x 106 cell/ml suspension. Then incubate your cells in the freezer (-20°C) for 5 to 7 minutes. Do not worry about keeping your cells sterile at this point- you are killing them.
Paraformaldehyde (cross-linking) method: In this method, paraformaldehyde is used to make chemical bonds (or cross-links) between the proteins in your cells and their surroundings. This method usually provides the best preservation, especially of soluble proteins. To fix by cross-linking, add an equal amount of 4% paraformaldehyde to your 2 x 106 cell/ml suspension to create a 1 x 106 cell/ml suspension. Then incubate your cells in this solution for 10 minutes at room temperature.
Step #5: Centrifuge your fixed suspension cells.
Now that you have a solution of fixed cells, you need to get these cells onto your cover slip. This is done using centrifugation. Place your clean cover slips in the buckets of a swing bucket centrifuge. (To keep your centrifuge sparkling clean, you may want to put some Whatman paper/blue roll down first to soak up any cell solution that may slide off your cover slips.) Then spot your cover slips with your fixed cell suspension. Spin at ~2,000 rpm for ~5 minutes. You now have fixed cells on your cover slips.
Step #6: Rinse.
Gently rinse your fixed cells with PBS* to remove any fixation agent. Be careful that you do not disrupt your cells when rinsing- pipette gently.
Step #7: Unmask if needed.
It you fixed using paraformaldehyde, you may need to unmask your antigen, because while cross-linking provides superior cell preservation it can also prevent or ‘mask’ your antibody from accessing your antigens. There are a variety of unmasking techniques out there. Most of which use some combination of proteinases, heat or chelators to make your cells more easily penetrated. See Catriona Paul’s article “Antigen Retrieval Techniques for Immunohistochemistry: Unmask that Antigen!” for more on unmasking your antigens.
Step #8: Stain and image.
When you’re done fixing, centrifuging, and unmasking, your cells are ready for staining and imaging when you are. However, if you are not ready just yet, your cover slips with their fixed cells can be stored under PBS in the refrigerator for up to three months.
Good luck and happy fixing!