Background
This is the method for indirect immunofluorescence labeling; that is, the antibodies do not have the fluorescent dye attached.Indirect labeling is more involved than direct labeling. If you need to label cells with different antibodies for simultaneous analysis, you may encounter problems choosing secondary antibodies (fluorochrome conjugated antibodies that bind to the primary antibody). For example, if both primary antibodies are mouse IgG 1, what can you do to label them individually with different fluorochrome conjugated secondary antibodies? (FITC-conjugated anti-Mouse IgG 1 will label both!) The biotin-avidin system may provide an answer, if one antibody can be biotinylated:
- label with the non-biotinylated mouse antibody first,
- then the FITC anti-mouse antibody,
- then the biotinylated mouse antibody,
- then PE-streptavidin.
OR, maybe one of the primary antibodies is available in a fluorochrome-conjugated form; incubate the cells with it after the primary and secondary antibody steps.
You can see how complicated this can become... The best solution for simultaneous labeling is get all direct conjugates, if at all possible. Then see our our protocol for direct labeling.
Materials
- Primary Antibodies:
- Test antibody: Usually mouse monoclonal IgG or IgM, not conjugated with any fluorescent dyes. For labeling cells with more than one test antibody simultaneously, you need primary antibodies made in different animals or have different Ig subclasses. Another option is to have on of them conjugated to biotin.
- Negative control sera: Purified protein (usually mouse IgM or IgG of the same subclass) to match your test antibodies.
In flow cytometry, fluorescence is relative. We need a negative control to determine where "positivity" begins.
- Secondary antibody: Fluorochrome-conjugated antibodies directed towards the protein composing the primary antibodies and negative control sera.
For simultaneous labeling, choose the fluorochromes carefully. You want the farthest red-emitting antibody combination to label the antigen with the greatest density (most receptors per cell).
- Fluorochrome-conjugated streptavidin: Use this if one of your primary antibodies is biotin conjugated.
- PBS with 0.1% sodium azide added. The sodium azide assists in preventing capping and shedding or internalization of the antibody-antigen complex after the antibodies bind to the receptors.
- Cells in suspension, counted and viability-checked. Keep them in culture medium supplemented with antibiotics and 2-5% fetal bovine serum, on ice. If viability is less than 90%, consider adding another fluorochrome to identify dead cells during analysis.
Equipment
- Centrifuge. You should know how the RPM translates into G-force.
- Precision adjustable micropipet. You will probably need two: one in the range of 10-100 microliters, and another ranging from 100-1000 microliters.
- Vortex mixer. You could mix by tapping or shaking the tubes, but a mixer will give much more reproducible results in most cases.
- 12x75 mm polystyrene tubes. The clear plastic kind. If you can, buy the Falcon brand because they fit the instrument best. If you can't, don't worry - we will supply them when you bring your samples to the lab.
- Ice bucket with cover. Generally, cells are more stable and tolerate insult better when they're cold. The cover keeps light out, which could bleach the fluorochromes.
- Flow cytometer. If you analyze your samples in our lab, the instrument you use will most likely be a FACScan or FACSort, made by Becton-Dickinson. (See our instrument list for more details.)
Procedure
- Adjust the cell concentration to 1 million per ml. with culture media or PBS.
- Place 1 ml. of the cell suspension into each of the 12x75 tubes.
You will need a tube for each primary and secondary antibody combination plus the negative control. If you're doing simultaneous labeling, use one tube for each combination of primary antibodies or controls, but we will need single-antibody labeled cells for each combination as well. Confused? Let's talk!
- Centrifuge at 250 x g for 5 minutes. Use a pipet to remove the liquid. Be careful not to disturb the pellet. A slight amount of liquid can remain.
This force and time works well for lymphoid cells. You may have to adjust as required if your cells are different.
- Add the appropriate amount of primary antibody or negative-control sera. The amount is usually given by the manufacturer. If not, it should have been determined previously by titration, using target cells with a large number of receptors.
- Vortex. Keep the tubes on ice for around 30 minutes. Cover the ice bucket.
- First wash - Add 1 ml. of the PBS+azide. Vortex.
- Centrifuge and remove liquid as above.
- Second wash - Add 1 ml. of the PBS+azide. Vortex.
- Centrifuge and remove liquid as above.
- Add the appropriate amount of secondary antibody or fluorochrome-conjugated streptavidin. The amount is usually given by the manufacturer. If not, it should have been determined previously by titration, using target cells with a large number of receptors.
- Vortex. Keep the tubes on ice for around 30 minutes. Cover the ice bucket.
- First wash - Add 1 ml. of the PBS+azide. Vortex.
- Centrifuge and remove liquid as above.
- Second wash - Add 1 ml. of the PBS+azide. Vortex.
- Centrifuge and remove liquid as above.
- Add 1 ml. of the PBS+azide. Vortex.
