Direct immunofluorescence labeling is labeling in which the antibodies of interest have the fluorescent dye already attached. Direct labeling is easier and faster than indirect labeling. Direct labeling is the preferred method if you are planning on labeling cells with two or more antibodies at the same time. That being the case there are situations in which the Indirect Method may be required or preferred, such as
- Absence of the antibody of interest in a conjugated form.
- Cell receptor number is low in which case the generally “brighter” labeling of the indirect method may allow better detection.
- Conjugation of the fluorochrome to the antibody interferes with the antibodies specificity.
- Fluorochrome-Conjugated Antibodies:
- Test antibody: Mouse monoclonal antibody conjugated to FITC, PE, PerCP etc. If you plan on labeling cells with two or more antibodies at the same time you need a negative control for each fluorochrome conjugate.
- Negative control sera: Usually, purified mouse IgM or IgG of the same subclass as the test antibodies and conjugated to the same fluorochrome. A negative control is necessary in order to distinguish between background autofluorescence and where a true positive signal exists.
- PBS with 0.1% sodium azide added. 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 which have been counted and assessed for viability. Cells should be kept in culture medium supplemented with antibiotics and 2-5% FBS, on ice. If viability is less than 90%, one should consider the uses of an additional label for live/dead discrimination.
- Adjust cell concentration to 1 million cells per ml.
- Place 1 ml of cell suspension into each of the 12x75 tubes (One tube for each antibody plus a negative control).
- Centrifuge at 250 x g for 5 minutes. Using a pipet remove the liquid being careful not to disturb the pellet. (Note: the above force and time is good for lymphoid cells. Adjustments may be necessary for different cell types).
- Add the appropriate amount of antibody to your tubes. Proper antibody concentration should be determined in advance via titration, using a known target cell with a large number of positive receptors.
- Vortex, and place cells on ice in covered bucket for 30 minutes.
- Wash cells by adding 1 ml of PBS w/ azide, vortexing, centrifuging and removing liquid as above.
- Repeat washing step for a second wash.
- After second wash and removal of liquid add 1 ml of the PBS w/ azide, vortex and place on ice until time for flow cytometric analysis.
Note: Lymphoid cells will generally last for several hours although that long a wait is generally not recommended. If you expect to be waiting that long or longer you should consider Fixing Your Cells which will preserve them for at least several days.