Basic Method for Direct Immunofluorescence Labeling

immunofluorescence labeling; that is, the antibodies have the fluorescent dye attached. Direct labeling is simpler and quicker than indirect labeling. We strongly recommend direct labeling if you're planning to simultaneously label cells with 2 or more antibodies. Still there are some reasons to go with the indirect method:

You cannot obtain your specific antibody in a fluorochrome-conjugated form;

Fluorochrome conjugation interferes with the specificity of the antibody;

The number of receptors per cell is low (indirect methods generally can render brighter labeling);

You need very accurate comparisons of labeling with different antibodies.

If any of the above apply, see our protocol for indirect labeling.

Materials

Fluorochrome-Conjugated Antibodies:

Test antibody: Mouse monoclonal conjugated to FITC, PE, PerCP or PE-CY5 Tandem (tradenames: Cy-Chrome or Tri-Color). If you're planning to label cells with 2 or more antibodies simultaneously, you need a negative control for each fluorochrome conjugate.

For simultaneous labeling, choose the fluorochromes carefully. You want the farthest red-emitting antibody to label the antigen with the greatest density (most receptors per cell).

Negative control sera: Usually, purified mouse IgM or IgG of the same subclass as the test antibodies and conjugated to the same fluorochrome. One usually purchases them from the same manufacturer to get a similar fluorochrome-to-protein ratio as the test antibodies.

In flow cytometry, fluorescence is relative. We need a negative control to determine where "positivity" begins.

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

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 antibody plus the negative control. If you're doing simultaneous labeling, use one tube for each combination of antibodies or controls, but we will probably 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 monoclonal antibody or 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 1 ml. of the PBS+azide. Vortex.

Keep the cells on ice, covered, until your scheduled time on the flow cytometer. Lymphoid cells will usually last for several hours, though it's not recommended to wait that long. If you anticipate waiting longer, consider fixing the cells, which can preserve them for at least several days or often longer.