scientist following Leinco's technical protocol for titration

Titration of Antibodies – Flow Cytometry Staining Protocol

Methods and Principles from our Scientific Staff


Why Do It?

The precise amount of an antibody is essential to proper titration.

Too much antibody will result in an increase in the background non-specific (or low affinity) binding, reducing the sensitivity of the fluorescent measurement.

Too little antibody will result in a decrease in the positive signal, again reducing the sensitivity of the fluorescent measurement.

Figure 1. (Left) As concentration of antibody increases there is greater separation of positive and negative cells based on fluorescence intensity.

Leinco Product C119, Anti-Human CD8 (Clone: UCHT-4) – FITC

How To Do It

Titration experiments are easy to perform, and there are several papers that discuss this process1,2. This protocol has been optimized for 1×106 cells in 50 µl final volume. All staining is performed on ice, in the dark, for 20 minutes. Identify the 1x concentration, which is the manufacturer’s recommended concentration for using their antibody in a given assay. These concentrations can either be a volume (10 µl per 100 µl) or a concentration (0.1µg/µl). Based on the starting concentration, generate a serial dilution series of 4 to 8 points. It is often good to include a 2x concentration to ensure the antibody is working well. If the optimal concentration is 2X the recommended usage, this can suggest a problem.

Solutions Needed

1. Staining Buffer: Phosphate Buffered Saline with 0.5% Bovine Serum Albumin (Fraction V) (See Leinco Part Number: F1175)
2. Fixation Buffer: 1-5% formaldehyde in PBS (See Leinco Part Number: F1194)
3. Blocking Buffer: 5-10% Normal (species) Serum in Staining Buffer. The species to use is based on the origins of the antibody being used. (Leinco has many Normal Serum species available).