Flow-Cytometric Immune Function Methodology(13)

Flow-Cytometric Immune Function 53

Fig. 6. Flow-cytometric results of TFN-α in LPS-activated CD11c– DCs. CD11c–DCs appear nonresponsive to LPS stimulation. Compare to Fig. 5.SeeFig. 4 for gating strategies. The uorescent parameters are displayed in a four-decade logarithmic scale.

CD11c+ DCs have low-scatter characteristics and highest expression levels of CD11c among all leukocytes. This gating strategy does exclude CD11c–events from the R1-gated HLA-DR vs lin 1 dot plot, resulting in improved cluster separation of CD11c+ DCs and non-DC leukocytes (1). Compare the resolution of the HLA-DR vs lin 1 dot plots in Fig. 3 (R1 gated) and Fig. 4(ungated). These plots are generated from the same data le (seeNote 4).3.4.2. CD11c– DC Gating Strategy (Fig. 4)

1. Create an HLA-DR vs lin 1 dot plot and display all events.

2. Draw a region R1 to include events with minimal lin 1 and highest HLA-DR

staining.

3. Create an HLA-DR vs CD11c dot plot that is gated on R1.

4. Draw a region R2 around CD11c– and HLA-DR-positive events. 5. Create a logical gate “G3 = R1 and R2” in the gate list. 6. Create a TNF-α vs CD11c dot plot that is gated on “G3 = R1 and R2.”See

Fig. 6.

For functional surface markers, this two-step gating strategy cannot suf- ciently resolve CD11c– DCs all the time (Subheading 3.1.3.). In these samples, the resolution of HLA-DR bright and lin 1 dim DCs is decreased from non-DC leukocytes. For this reason, we do not recommend this gating strategy for the inexperienced user. Instead, the reader can use CD123 as a positive identi cation marker in combination with HLA-DR and lin 1. This requires the processing of an additional sample set (Subheading 3.2.2.), because CD11c+ DCs cannot be reliably identi ed by the use of CD123, HLA-DR, and lin 1 cocktail (seeNote 4

).