Why is it Important to Consider Antibody Titration When Designing Flow Cytometry Experiments?

The importance of antibody titration

Antibodies are widely used for scientific, clinical and commercial purposes. But poorly validated antibodies and their batch-to-batch variability can create difficulties during experiments.^1,2 This can lead to abandoned projects and wasted time, money and samples. It is also considered the major driver of the ‘reproducibility crisis,’ a growing realization that many biomedical experimental results cannot be reproduced.²

Improper antibody dilution can generate unwanted background and is one of the major sources of low-quality results in flow cytometry.³ Unfortunately, the vendor-recommended dilution is not always appropriate for the specific assay conditions.³

Low antibody concentrations can make a positive population appear negative, while high antibody concentrations can make a negative population appear positive.⁴

To overcome this, it is important to consider antibody titration when designing multicolor flow cytometry panels.⁵

Antibody titration is necessary to optimize reagent performance and ensure adequate separation between the positive population and background signal.⁶

The optimal antibody concentration is determined by the maximum stain index of the antibody.^5,6 It is also influenced by several factors such as staining temperature and duration, as well as the type of fixation/permeabilization buffer. Therefore, it is recommended to titrate antibodies under the same conditions as the final experiment.⁶
 

Titrations should be conducted and repeated for each antibody in the panel to ensure the accuracy and reproducibility of the titration experiment.⁵

Protocol to establish optimal antibody concentration ⁷

As well as your centrifuge, flow cytometer and round-bottom tubes, you will need:

• — Antibodies of interest labeled with a fluorochrome (at a concentration 4x the manufacturer’s recommendation; can be higher or lower if desired)
• — Phosphate buffered saline without calcium or magnesium (containing 1% bovine serum albumin)
• — Sample: cell suspension (1–5 × 10⁶ cells/mL); the cells should be prepared, unfixed or fixed, in the same way as they will be for the multicolor experiment

Prepare antibody serial dilutions

• 1.  Label each of your tubes from 1 to 9.
• 2.  Add 50 µL of staining buffer to each tube.
• 3.  Add 50 µL of antibody at 4x the manufacturer’s recommendation to the first tube.
• 4.  Mix well and transfer 50 µL from tube 1 to tube 2.
• 5.  Repeat this step until tube 7 contains 50 µL, then discard this from tube 7.

Add cells

The cell preparation must contain a mix of cells that are positive and negative for the epitope.

• 1. Add 100 µL of the 1–5 × 10⁶ cell/mL suspension to all tubes and mix well.
• 2. Incubate for 30 minutes at room temperature in the dark (or follow recommended conditions for your antibody).
• 3. Wash the cells three times by adding 2 mL of staining buffer and centrifuge for 5 minutes at 300 x g, 4 °C. Remove the supernatant, vortex the pellet and resuspend in 200 µL of staining buffer.

After flow cytometry data analysis, a titration curve can be constructed to compare the stain index to antibody concentrations; the maximum stain index indicates optimal antibody concentration.⁵

Warning: if antibody affinity is low, the titration curve will have no clear saturation plateau, meaning the optimal antibody concentration is difficult to obtain. In this case, the experiment may be prone to false-negative or false-positive results.³

Discover more flow cytometry protocols.