Flow-Cytometric Immune Function Methodology(9)

Flow-Cytometric Immune Function 49

For DCs, the acquisition gate is de

(seeFig. 2 ned carefully because any event outside of it will not be recorded in the data le. For rare-cell populations, the data analysis can be inef cient without the use of live gating during sample acquisition because large data les (>50,000 events) signi cantly slow down the speed of data processing. Use the entire cell suspension of each sample for acquisition, which yields around 5000 to 8000 events. It might be helpful to prepare an additional sample to perform the FSC threshold adjustment and to de ne the acquisition gate.3.4. Data Analysis

Dendritic cells can be characterized by low FSC and side scatter (SSC), high expression levels of HLA-DR, and lack of or minimal staining for lineage markers. Two different gating strategies have been developed for CD11c+ and CD11c– DCs (seeFigs. 3 and 4). CD11c+ DCs respond to LPS stimulation, as shown by the detection of increased levels of TNF-α, CD80, and CD83, whereas CD11c– DCs remain unresponsive for TNF-α (seeFigs. 5 and 6)(seeNote 4).

3.4.1. CD11c+ DC Gating Strategy (Fig. 3)

1. Create an SSC vs CD11c dot plot and display all events of the data le.

2. Draw a region R1 to include CD11c bright and SSC low events as demonstrated

inFig. 3. The drawing of region R1 is not based on a discrete cluster resolution.