Flow-Cytometric Immune Function Methodology(7)

Flow-Cytometric Immune Function 47

17. Centrifuge 10 min at 500g. 18. Decant the supernatant.

19. Resuspend the now intracellular-stained and permeabilized cells in approx 200 μL

wash buffer.

20. Acquire the samples in ≤1 h on the ow cytometer.

21. Store samples at 4°C if not acquired immediately (seeNote 2).

3.2.2. Sample Processing: Functional Surface Antigen Detection

Sample processing for functional surface antigen detection includes cell surface staining with uorophore-conjugated monoclonal antibodies followed by red blood cell lysis. The protocol uses activated and resting samples from the procedure in Subheading 3.1.3.

1. Add 60 μL lin 1 FITC, 30 μL anti-HLA-DR PerCP, 15 μL CD11c APC, and

20μL CD83 PE to an empty 5-mL polypropylene round-bottom tube. 2. Add 60 μL lin 1 FITC, 30 μL anti-HLA-DR PerCP, 15 μL CD11c APC, and

60μL CD80 PE to a second empty 5-mL polypropylene round-bottom tube. 3. The following processing steps apply to both sample tubes: a. Add 300 μL WB to the antibody mixture and vortex. b. Incubate for 15 min at RT in the dark. c. Add 3 mL of FACS Lysing Solution 1X working concentration; cap tubes

and vortex.

d. Incubate for 10 min at RT in the dark. e. Centrifuge 7 min at 500g. f. Aspirate the supernatant and vortex to break off the cell pellet. g. Add 2 mL wash buffer and vortex. h. Centrifuge 7 min at 500g. i. Aspirate the supernatant. j. Resuspend stained cells in approx 200 μL of wash buffer. k. Acquire the samples in ≤1 h on a ow cytometer. l. Store samples at 4°C if not acquired immediately (seeNote 2).

3.3. Flow Cytometry Protocols3.3.1. Instrument Calibration

The ow cytometer is set up for acquisition of uorescently stained blood samples, derived from Subheading 3.2., with CaliBRITE beads and FACS-Comp software. The four-color bead system is composed of unlabeled beads and FITC-, PE-, PerCP-, and APC-labeled beads that mimic auto uorescence of leukocytes and uorescence emission of stained cells. Beads and software provide a system that automatically adjusts the instrument sensitivity and corrects for spectral overlap of the fluorescent dye emissions, known as uorescent compensation, respectively.