What are the appropriate cell and antibody amounts for flow cytometry antibody titration?

For titration, it is recommended to fix the cell number at 1×10^6 and use a staining volume of 100 μL for greater stability, because too small a volume leads to high local antibody concentration and large mixing errors. Start antibody titration at 2 μg/test with 2-fold serial dilutions, generally down to 0.03–0.06 μg/test, covering 7–8 points; more points are of little value. Recommended amounts vary greatly among brands, mainly depending on the clone number and degree of fluorescent labeling, so do not simply follow the recommendations in the instructions. To determine the optimal concentration, evaluate the staining index or signal-to-noise ratio of the positive and negative populations, and choose the lowest concentration on the plateau phase.

For human PBMC surface staining, Fc block is recommended, especially for samples with abundant monocytes/macrophages or B cells, as Fc receptor capture of antibodies can cause false positives. Commercial human FcR blocking reagent, 5-10% homologous serum, or 2% BSA can be used, but note that the concentration and duration of Fc block must be kept consistent between titration and formal experiments. If the budget is limited, 5% FBS/PBS blocking solution can also be used as a substitute, but do not omit this step; after blocking, there is no need to wash before directly adding antibodies for staining. If concerned about affecting staining, blocking can be performed for 10 minutes.

It is normal for different brands of antibodies to recommend different usage amounts. Even for the same CD antibody, differences in clone number, fluorochrome, and F/P ratio can result in several-fold differences in the actual effective concentration. The core of titration is not to rely on the brand's recommendation, but to use your own staining system to find the plateau phase. It is recommended to test two lots of the antibody. If the laboratory already has antibodies of the same fluorochrome from other brands, a side-by-side comparison can be performed first. Record the MFI and negative SD. The optimal concentration is usually at the lowest point where the signal-to-noise ratio no longer increases significantly. After titration is complete, aliquot into small volumes and store protected from light to avoid repeated freeze-thaw cycles.