Flow-Cytometric Immune Function Methodology(5)

Flow-Cytometric Immune Function 45concentration. The baseline of TNF-α expression is determined with a resting control that contains BFA but lacks the stimulus LPS. Instead, stimulus solvent is added to balance the concentration of DMSO in activated and resting samples. Activated and resting samples are incubated in parallel.

Prepare activated control:

Add 1 mL of whole human blood to a 5-mL polypropylene round-bottom tube. Add 20 μL of LPS working solution (0.05 mg/mL) to the same blood and vortex. Add 20 μL of BFA working solution (0.5 mg/mL) to the same blood and vortex. Incubate for 2 h.

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. Add 20 μL of BFA working solution (0.5 mg/mL) to the same blood and vortex. Incubate for 2 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.

3.1.2. Blood Activation: Cytokine Kinetic Assay (TNF-αβ, IL-6)

processed every hour in a time window from 0 to 8 h of incubation. Follow the basic procedure described in Subheading 3.1.1. for the positive activation control(1) with one variation: BFA is added to whole blood during only the last hour of sample incubation. This determines the cytokine production in 1-h time increments. Each time-point requires the preparation of one activated whole-blood sample.

3.1.3. Blood Activation: Functional Surface Antigen Detection (CD80, CD83)

Peripheral blood is activated with LPS at 1 μg/mL nal concentration in the absence of secretion inhibitor BFA. The baseline of CD80 and CD83 expression is determined with a resting control that lacks LPS but contains stimulus solvent to balance the concentration of DMSO in activated and resting samples. Activated and resting samples are incubated in parallel.

Prepare activated control:

Add 1 mL of whole human blood to a 5-mL polypropylene round-bottom tube. Add 20 μL of LPS working solution (0.05 mg/mL) to the same blood and vortex. Incubate for 4 h.