Flow-Cytometric Immune Function Methodology(4)

44 Willmann

4. FACS Lysing Solution (10X) from BD Biosciences (San Jose, CA, cat. no.

349202). Make a 1X working dilution with deionized water. Store at room temperature (RT).

5. FACS Permeabilizing Solution 2 (10X) from BD Biosciences (San Jose, CA) cat.

no. 340973). Make a 1X working dilution with deionized water. Store at RT. 6. Wash buffer (1X PBS with 0.5% bovine serum albumin [BSA]).

7. Falcon 5-mL polypropylene round-bottom tubes with caps (12 × 75-mm style)

from BD Discovery Labware (Bedford, MA, cat. no. 352063).

8. Falcon 5-mL polystyrene round-bottom tubes (12 × 75-mm style) from BD

Discovery Labware (Bedford, MA, cat. no. 352052). 9. Sorvall benchtop centrifuge. 10. Vortex.

2.3. Flow-Cytometric Sample Acquisition

The following components are a complementary system that is speci cally designed for the multicolor analysis of uoresceine isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophyll protein (PerCP), and allophycocyanin (APC). Other uorophore combinations cannot be used because they have alternate system requirements.

1. FACSCalibur dual-laser ow cytometer.

2. FACSComp software for instrument setup.

3. CellQuest or CellQuest Pro software. 4. CaliBRITE 3 beads, unlabeled, FITC-, PE-, PerCP-labeled for instrument

setup (cat. no. 340486).

5. CaliBRITE APC beads for instrument setup (cat. no. 340487).

All products are distributed by BD Biosciences (San Jose, CA).2.4. Flow-Cytometric Data Analysis

CellQuest, CellQuest Pro, or PAINT-A-GATE Pro software from BD Biosciences (San Jose, CA).3. Method

3.1. Blood Activation Protocols

Collect venous blood of normal donors in sodium heparin VACUTAINER tubes. Other anticoagulants severely compromise the functional capacity of lymphocytes and are not recommended (seeNote 1).

3.1.1. Blood Activation: Intracellular Cytokine Detection (TNF-α)

Peripheral blood is activated with LPS at 1 μg/mL final concentration in the presence of secretion inhibitor Brefeldin A (BFA) at 10 μg/mL nal