Acquisition and Analysis

All samples were analyzed on a FACS Caliburâ„¢ using the software CellQuest (BD Biosciences). After launching the acquisition software, all instrument parameters have to be adjusted for the cells you want to analyze. One has to make sure that the instrument is compensated properly (see Note 10).

The PE-stained beads are run first, at least 10,000 events should be acquired. Place an active gate on the bead singlet population using forward scatter (FSC) and side scatter (SSC). The instrument setting should remain unchanged with respect to fluorescence once the beads have been run. The analysis of the samples should be performed using the same settings.

1. Reconstitute one tube of QuantiBRITE beads with 0.5 mL of PBS and vortex. Run the QuantiBRITE bead tube thresholding on FSC or SSC and collect 10,000 events. Display a FSC vs SSC dot plot and adjust the gate around the singlet bead population.

2. Display a FL-2 histogram and adjust markers around the four bead populations. View histogram statistics (make sure geometric means are displayed).

3. Click the "Copy means" button to copy the geometric means of the four bead peaks from the histogram statistics window.

4. Select histogram statistics view and choose quantitative calibration from the "Acquire" menu. Enter the lot-specific PE/bead values from the flyer in the kit.

5. Click "Calibrate" for CellQuest to perform regression analysis and to display the slope, intercept, and correlation coefficient. Save the document.

6. Do not adjust photomultipliers or compensation after acquiring these events.

3.3.2. Acquisition and Analysis of Patient Samples

1. Display a FL-1 vs FL-3 dot plot and gate on monocytes (Fig. 1A). Count at least 3000 gated events in each sample (see Note 11).

2. Display a FL-2 histogram of the monocytes. The geometric mean of the entire monocyte population is then used to determine the ABC.

3. Granulocytes were recognized in the forward vs sideward scatter diagram (Fig. 1B).

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