How do you titrate a flow cytometry antibody and pick the concentration to use?

Why

The vendor's recommended amount was set on someone else's cells, buffer, time and temperature. The Cossarizza guidelines therefore recommend titrating every reagent, and every new lot, in the conditions of the actual experiment. Separation is measured on both populations.

The stain index compares the gap between positive and negative medians with the spread of the negatives. It falls at high concentrations because the negatives brighten, and at low ones because the positives dim. A population that lacks the antigen is needed in the sample to read background.

Concentration, not total amount, is the key variable, so scaling up the staining volume needs proportionally more antibody. Staining is robust within a fairly wide range of cell numbers. Fixed and live cells, or surface and intracellular staining, can have different optima.

How to work it through

1. Use the same cell type, cell number, buffer, volume, time and temperature as the real assay.
2. Start from the vendor's concentration or a generic high point and make six to eight twofold dilutions.
3. Include unstained cells and make sure a negative population is present.
4. Acquire all tubes with identical instrument settings.
5. Plot the median of positives, the median of negatives and the stain index against concentration.
6. Choose the concentration with the best separation, or the lowest one that reaches near-saturating signal if robustness matters more.

Where the answer stops holding

An optimum found in a single-color tube can shift inside a full panel, where spread from other dyes changes the negative population. Titration on a cell line may not hold for primary tissue with different antigen density. Very rare targets may need saturating staining for consistency across samples, even at some cost in background.