Flow-Cytometric Immune Function Methodology(3)

Flow-Cytometric Immune Function 432. Materials

The immune function methodology has been optimized in great detail with the following materials. It is unknown if similar products provide the same results with the protocols described in Subheading 3.2.1. Blood Activation

1. B VACUTAINER® tubes with sodium heparin for blood collection (sterile

12× 75-mm style) from BD Discovery Labware (Bedford, MA, cat. no. 367671). 2. Falcon® 5-mL polypropylene round-bottom tubes with caps (12 × 75-mm style)

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

3. Dimethyl sulfoxide (DMSO) from Sigma (St. Louis, MO, cat. no. D-8779).

4. LPS from Sigma (St. Louis, MO, cat. no. L-2654). Make an LPS stock solution

of 0.5 mg/mL in DMSO and store 20-μL aliquots at –70°C. LPS dissolves easily in DMSO at room temperature. To make an LPS working solution, thaw a 20-μLaliquot of the 0.5 mg/mL LPS stock solution and dilute it 1 10 in 1X phosphate-buffered saline (PBS) to achieve a nal working concentration of 0.05 mg/mL. Use this solution within 1 h.

5. Brefeldin A (BFA) from Sigma (St. Louis, MO, cat. no. B-7651). Make a BFA

stock solution of 50 mg/mL in DMSO and store at –70°C. To make a BFA working solution, thaw the 50 mg/mL BFA stock solution and dilute a 5-μLaliquot 1 100 in 1X PBS to achieve a nal working concentration of 0.5 mg/mL. Use this solution within 1 h.

6. Phosphate buffer saline (PBS) 1X. 7. Vortex.

2.2. Sample Processing

1. Lineage cocktail 1 (lin 1) FITC from BD Biosciences (San Jose, CA, cat.

no. 340546). The lineage cocktail contains a mixture of antibodies that stain lymphocytes, monocytes, eosinophils, and neutrophils. Lin 1 includes antibodies to CD3, CD14, CD16, CD19, CD20, and CD56. The use of CD19 and CD20 in parallel provides a brighter staining of B-cells with lin 1. Peripheral blood dendritic cells and basophils can be distinguished from other leukocytes by their lack of staining with lin 1 FITC. The lin 1 antibody cocktail is provided at working concentration. Store the monoclonal antibody reagent at 2–8°C in the dark.

2. Anti-HLA-DR peridinin chlorophyll protein (PerCP), 0.0125 mg/mL (cat. no.

347364); CD11c allophycocyanin (APC), 0.025 mg/mL (cat. no. 340544);CD80 phycoerythrin (PE), 0.0032 mg/mL (cat. no. 340294); anti-TNF-α PE, 0.0032 mg/mL (cat. no. 340512); anti-IL-1-β PE, 0.003 mg/mL (cat. no. 340516); anti-IL-6 PE, 0.0015 mg/mL (cat. no. 340527); all from BD Biosciences, San Jose, CA. All monoclonal antibody solutions are provided at working concentra-tion. Store reagents at 2–8°C in the dark.

3. CD83 PE custom product, working solution 0.05 mg/mL, from BD Biosciences

(San Jose, CA). Store monoclonal antibody reagent at 2–8°C in the dark.