Titration Example

Tutorial for Titrations

When undertaking flow cytometeric analysis of cells labelled with fluorescent antibodies against a cell surface marker it is often advisable to titrate the amount of antibody being used. Some of the reasons why you would do this are:

If you use too little antibody,the staining will not be sufficiently bright to detect the cells with bound antibody from background staining.

If you use too much antibody, the fluorescence may be so great that the signal falls well out in the detection range and the signal is compressed due the nature of the logrithmic amplifier, thus it is inherently more inaccurate.

If you use too much commercial antibody it can be expensive, and often a lower concentration of antibody will give an identical percentage positively labelled cells with sufficient separation of signal from background to be usable. Remember, commercial antibody companies want you to buy more of their product... The best method of understanding this is often to look at an example and note the median (compare with mean) fluorescence values. As the antibody concentration present in the serum decreases, the median fluorescence intensity decreases as the peaks shift to the left.

The example below shows a titration of serum containing antibodies against human endothelial cells.

Please move mouse over histogram peaks please!
Clicking near the top of a "peak" will provide further information.