How to Perform Antibody Titration？

The concentration of antibodies is a critical parameter when using Flow Cytometry. Adding antibodies arbitrarily can result in problems such as excessive background staining and sample wastage, especially for novice users. In order to achieve better experimental results and conserve precious samples, antibody titration is necessary.

1. What is antibody titration?

Antibody titration is an experiment conducted before performing flow cytometry experiments to determine the optimal antibody dosage and concentration for the antibodies used. Its purpose is to achieve the best signal-to-noise ratio and play an important role in antibody staining for multi-parameter flow cytometry experiments.

2. Why is antibody titration necessary?

Generally, the manufacturer provides recommended usage amounts for flow cytometry antibodies. However, the optimal concentration or "titer" of antibodies depends on the experimental conditions, such as the detection target, staining time, temperature, and whether the cells are fixed.

As commercial reagents are unlikely to be tested under your specific experimental conditions, using too much or too little antibody can result in increased non-specific staining, decreased signal-to-noise ratio, decreased sensitivity, and lack of linear relationship between expression levels and staining intensity, which increases experimental costs. Therefore, antibody titration is particularly important.

3. How to perform antibody titration

① Before using the antibody (taking mouse CD4 as an example), centrifuge at 12,000 g for 1 min to remove bottom precipitates, which may be formed by antibody aggregation.

② Generally, start with a dilution of about 10 μg/mL and make 6-8 gradients for titration. For example, if the initial concentration of the antibody is 0.2 μg/μL and the experimental system is 100 μL, make 6 gradient concentrations:

perform antibody titration

Tube 1: 10 μL antibody + 90 μL dilution buffer

Tube 2: Take 50 μL from Tube 1 + 50 μL dilution buffer

Tube 3: Take 50 μL from Tube 2 + 50 μL dilution buffer

And so on

Tube 6: Take 50 μL from Tube 5 + 50 μL dilution buffer, mix well and discard 50 μL of the antibody

Tube 7: Add only 50 μL dilution buffer for blank control

In this way, you only use 2 μg of antibody for this titration.

③ Pre-block the cells to be labeled with an appropriate amount of Fc receptor blocking reagent at room temperature for 10 min.

④ Add 50 μL of approximately 1×10⁶ cells to each tube of diluted antibody, mix well, and incubate at 4°C in the dark for 30 min.

⑤ Add 2 mL of cell staining buffer, gently mix, and centrifuge at 300 g for 5 min.

⑥ Discard the supernatant, resuspend in 200 μL PBS, and analyze on the flow cytometer. Acquire at least 500 events in the positive cell population.

4. How to Calculate Titration Results

Once the titration gradient is established, there are several methods to evaluate the data to determine the optimal titration:

The simplest method is to calculate the Signal-to-Noise Ratio (SNR), which is the ratio of the Median Fluorescence Intensity (MFI) of the positive population to the MFI of the negative population. As shown in the figure, the optimal antibody concentration for CD4-FITC is 0.7 μg/mL (1:800 dilution).

Calculate Titration Results

▲Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol. 2019 Oct; 49(10): 1457–1973.

Considering the coefficient of variation (CV) of the negative population, another method is to calculate the Staining Index (SI):

calculate the Staining Index

As shown in the figure, the staining index is maximized at an antibody concentration of 1.25 μg/mL for CD4-PE, which is the optimal concentration for use.

5. Considerations for Antibody Titration

① For some fully positive cells, unstained cells can be added after sample staining during titration.

② For markers with distinct subpopulations and large intervals, such as CD3, CD4, CD8, titration may not be necessary when setting gates for cell subset identification, only for markers that require analysis of proportion or MFI.

③ For rare cell populations with low expression, antibodies for the parent cell population can be added during staining.

④ If there are a large number of dead cells in the sample, dead/live staining should be performed first during titration to distinguish dead and live cells, and then the optimal concentration of the target antibody should be determined.

⑤ Within a certain range of cell density, the staining index of the antibody is only related to the concentration of the antibody and not related to cell density.

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