Calculating a Single Concentration
Contents
To set up the proper parameters for the concentration calculator, select the View tab→Tools→Concentration Calculator command.
To calculate a single concentration:
| • | Go to the Individual tab of the Concentration Calculator dialog (Figure 13.1). |
Figure 13.1 Concentration Calculator - Individual Tab
| • | Use the File to analyze drop-down list box to select the FCS file on which you want to calculate the concentration. You can choose to calculate the concentration for only a subset of cells by selecting a gate in the Gate to analyze list box. |
| • | Select a method to calculate volume using Reference beads or acquisition Time (see below). |
| • | Press Calculate to calculate the actual concentration. The sample volume will be displayed in the Volume Analyzed field and the concentration will be displayed in the Sample Concentration field. |
| • | Calculating Volume from Reference Beads |
A common way to calculate the volume of a sample is to add a known concentration of beads to your sample. By counting how many beads were acquired in the time it took to acquire the sample, it is possible to calculate the volume of the sample.
| o | Before opening the Concentration Calculator dialog, draw a gate around your population of beads on a 2D Plot. |
| o | Select the Reference Beads radio button |
| o | Enter the gate number in the Bead Gate field. |
| o | Enter the concentration of the beads in the Bead Concentration field. The concentration must be entered in units of beads/ml. You can enter the concentration in scientific notation (e.g., 5 x 106/ml is entered as 5e6). |
| • | Calculating Volume by Time |
Some instrument sheath fluid flows at a well-defined rate. If you know the flow rate, and the acquisition time, you can calculate the volume acquired.
| o | Enter the flow rate in the Flow Rate field in ml/min. |
| o | The Start Time and End Time fields are automatically filled in from data in the topmost FCS file header. If those times are not correct, you can enter the start time and end time directly. The Total Time is calculated as the difference between the start time and end time. You can also enter data into the total time field directly. |