scientist following Leinco's technical protocol for titration

Titration of Antibodies – Flow Cytometry Staining Protocol

Methods and Principles from our Scientific Staff

Background

Titration is the process of identifying the correct concentration of antibody to use for a given assay. This ensures the antibody performs within acceptable parameters including reducing negative effects and waste. Titration for flow cytometry is used to determine the antibody amount and concentration resulting in the highest signal of the positive population along with the lowest signal of the negative population. It is also the best way to eliminate nonspecific binding of an antibody.

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 process^1,2. 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).

Protocol (using 12x75 mm tubes)

Prepare cells at a final concentration of 2.5×10⁴ per µl in blocking buffer
Aliquot 40 µl of cells per sample
1. If using a viability dye, include a single stained control for viability (for compensation)
Prepare the dilution series
1. Prepare the concentration so that 10 µl can be added to each sample
Add the antibody to the cells and gently mix
Place on ice, in the dark, for 20 minutes
Add 3 ml staining buffer to the sample
Centrifuge the sample at 900xg
Aspirate the solution while not disturbing the pellet
Resuspend the sample in ~300 µl final volume
1. If using viability dye, add to the sample
2. If sample has to be fixed, resuspend in~300 µl of fixation buffer
  1. If using fixation buffer, cell impermeant viability dyes (PI, 7AAD, DAPI, etc) cannot be used
Analyze the cells on the flow cytometer
1. Collect sufficient events for analysis (10,000 positive events)
Export the data and analyze in software of choice
Calculate the Staining Index (SI) and plot versus concentration of antibody³
1. Staining Index (SI) = ((medianpos-medianneg)/((84%neg-medianneg)*0.995)

Titration of Antibodies – The Basic Flow Cytometry Staining Protocol