In the course of this example, we will:

•Identify the variables needed for the concentration calculation.

•Set up the concentration calculator using beads.

•Obtain a one time result.

In order to calculate the concentration, we need the following four parameters:

A.The file to be analyzed.

B.The cells of interest.

C.The gate containing the beads that were added to the sample tube.

D.The concentration of the beads in the sample tube.

Now, the gating strategy for the cells and beads is ready, we will proceed to do the actual calculations. Note this and the following sections continue from steps 1 through 7 in the Setting up the Beads Gates chapter.

1.Select the Tools tab→Calculate group→Concentration Calculator command .

The Concentration Calculator dialog now appears as shown in Figure T24.3.

2.Select the File to analyze, in this case Monocytes.002, from the drop-down list (Figure T24.3).

3.Select Monocytes as the Gate to analyze, from the drop-down list (Figure T24.3, ).

4.Select the Reference Beads radio button to specify the Volume Calculation type (Figure T24.3, ).

5.Select Beads2 for the Bead Gate (Figure T24.3,).

6.Input the concentration of the beads in the sample tube. In this case we will use a value of 100,000 beads/ml (Figure T24.3, ).

7.Click the Calculate button.

In the Results section of the Concentration Calculator, the volume of the sample analyzed appears in the Volume Analyzed (Figure T24.3,) field and the concentration of the cells in the Monocytes gate appears in the Sample Concentration field (Figure T24.3, ).

 

8.Click Cancel to close the Concentration Calculator dialog.

Note:  The volume and concentration values may differ due to gate placements.  Minor variances in the numerical values that are represented in the tutorial images are expected. Please save this layout to your desktop if unable to complete the next tutorial section.

In the next example, we will calculate the concentration using time and flow rate.