Flow-Cytometric Immune Function Methodology(6)

46 Willmann

Prepare resting control:

Add 1 mL of whole human blood to a 5-mL polypropylene round-bottom tube. Add 20 μL of DMSO diluted 1 ; 10 in PBS 1X to the same blood and vortex. Incubate for 4 h.

The cell activation is performed at 37°C humidi ed atmosphere and 5% CO2. During incubation, cap tubes loosely to prevent evaporation of the sample, but to allow gas exchange. The use of polypropylene labware for DC activation is important because it increases the DC recovery. DCs can stick to polystyrene surfaces and may be lost for further analysis.3.2. Sample Processing Protocols

The activated and resting control samples are processed in parallel for acquisition by ow cytometry. All steps of the procedure are performed at RT and in polypropylene tubes.

3.2.1. Sample Processing: Intracellular Cytokine Detection

Sample processing for intracellular antigens includes surface staining with uorophore-conjugated antibodies, followed by red blood cell lysis, cell permeabilization, and intracellular cytokine staining using uorophore-conjugated antibody. This protocol uses activated and resting samples from the procedure in Subheadings 3.1.1. and 3.1.2.

1. Add 120 μL lin 1 FITC, 60 μL anti-HLA-DR PerCP, and 30 μL CD11c APC to

an empty 5-mL polypropylene round-bottom tube. 2. Add 300 μL whole blood (WB) to the antibody mixture and vortex. 3. Incubate for 15 min at RT in the dark.

4. Add 3 mL FACS Lysing Solution 1X working concentration, cap tubes, and

vortex.

5. Incubate for 10 min at RT in the dark. 6. Centrifuge 7 min at 500g.

7. Aspirate supernatant carefully and vortex gently to break off the cell pellet.

8. Resuspend surface-stained cells in 1 mL FACS Permeabilizing Solution 1X

working concentration.

9. Incubate for 10 min at RT in the dark.

10. Add 2.5 mL wash buffer, cap tubes, and vortex gently. 11. Centrifuge 10 min at 500g.

12. Decant the supernatant instead of aspirating.

13. Resuspend permeabilized cells in remaining supernatant of the 5-mL tube

gently. 14. Add 20 μL anticytokine PE reagent for intracellular staining. 15. Incubate for 30 min at RT in the dark. 16. Add 2 mL wash buffer and vortex gently.