Flow-Cytometric Immune Function Methodology(11)

Flow-Cytometric Immune Function 51

Fig. 4. Data analysis. Identi cation of CD11c– in LPS-activated blood. The rst row shows LPS-activated samples and the second row displays resting peripheral blood. CD11c– DCs are de ned by their high HLA-DR expression and their lack of lineage markers and CD11c antigen. The left plot is ungated. The right plot is gated on R1. The uorescent parameters are displayed in a four-decade logarithmic scale.

3. Create an HLA-DR vs lin 1 dot plot that is gated on R1.

4. Go back to the SSC vs CD11c plot. Now, adjust the position and size of region

R1 while observing the changing cluster separation of the HLA-DR bright lin 1 dim events in the R1 gated HLA-DR vs lin 1 dot plot. Choose the nal de nition for region R1 that re ects the best cluster resolution for the brightest HLA-DR and least lin-1-stained event.

5. Draw a region R2 around the cell cluster with minimal lin 1 and highest HLA-DR

staining.

6. Create a logical gate “G3 = R1 and R2” in the gate list. 7. Create a TNF-α vs CD11c dot plot, a CD80 vs CD11c dot plot, and a CD83 vs

CD11c dot plot that are each gated on “G3 = R1 and R2.”SeeFig. 5. Additionally, you can create a TNF-α

histogram that is gated on “G3 = R1 and R2.”