Flow-Cytometric Immune Function Methodology(8)

48 Willmann

1. Freshly prepare one tube with 1 mL of sheath uid or wash buffer and a second

tube with 3 mL of sheath uid or wash buffer.

2. Then, add one drop of unlabeled beads and APC-labeled beads to the rst tube

and all ve beads, unlabeled—FITC, PE, PerCP, APC—to the second tube.

3. Vortex and follow the FACSComp software instructions on the computer that

is connected to the cytometer.

4. Manual instrument setup procedures are feasible but have not been validated for

the DC immune function protocols.

3.3.2. Sample Acquisition

A ow cytometer counts particles, including cellular debris, as well as leukocytes of interest. It is the goal to include as little cellular debris as possible and to maximize the cell count in every data le. The forward scatter (FSC) parameter is a relative measure for particle size and, optimally adjusted, it reduces the number of small debris events in the data les. For this reason, we recommend ne-tuning the FSC threshold prior to sample acquisition (Fig. 1).The threshold setting might need to be readjusted when a different set of samples is run (i.e., LPS vs LPS and BFA-treated samples).

Dendritic cells are found at low frequency in peripheral blood. Therefore, many cells (events) have to pass through a ow cytometer to collect a statisti-cally significant number of DCs (seeNote 3). The more events that are recorded, the more space a data le takes for storage. Therefore, many ow-cytometric investigators choose live gating as an option in rare-event analysis during sample acquisition. The use of an acquisition (live) gate reduces the number of irrelevant leukocytes over the number of desired DCs in a data

le.