Select a sample that you want the number of molecules for.
Copy the derived parameter to the All Samples group. (An a/b symbol appears beneath your sample.) Derive Parameters window, showing the parameter definition. x is the parameter being used to measure the number of molecules, andįigure 8. The derived parameter should equal the definition of a line, y = mx + b, where: Enter the slope of the line from Step 19.Ĭlick the “+” button, and add the intercept from Step 19. Click the Multiply button, or add an asterisk to the nascent expression. Select the parameter used for the calibration (for example, FITC). Just below the plot, in the formula panel, click Insert Reference. In the Derive Parameters menu, enter a name for the parameter (for example, the No. From the drop-down menu, select Derive Parameters. In the workspace, right-click on a sample. Correlation Plot, showing slope and intercept. (You can save the image, or leave the plot open.)įigure 7. Note the slope of the line and the intercept. (If they’re reversed, simply click Transpose Axes.) Ensure the target fluorochrome is on the X-axis and the “No. In the Plots band, click the Correlation Plot button. In the Table Editor, highlight both entries. Table Editor, showing the original and new entry. The Table Editor should now have two entries, the MFI statistic and the “No. Add Column dialog, showing the File Keywords pane. Select the keyword you added in Step 2 from the list of keywords in the left pane, and click OK.įigure 5. Add Column dialog, showing the Keyword tab. In the Add Column dialog window, click the Keyword tab.įigure 4. From the Columns band, select Add Column. Drag in the MFI statistic node into the Table Editor. Add the median or geometric mean statistic (MFI) to one of the gated populations, and copy it to the group.įigure 3. Move the ranged gates in the remaining samples to their appropriate positions. Copy the gate to the group (Command + Control + Shift + G). Graph window, showing a ranged gate on the histogram’s modal population. Create a ranged gate on the modal (peak) population.įigure 2. Change the plot to a histogram with the primary channel on the X-axis. Open the sample representing the calibration blank. (These should be known values provided by the manufacturer, for example 8,000, 16,000, 64,000, and so on.) In the workspace, add the appropriate values to the “No. Sample window, showing new keyword column. (Note: If you have a keyword/value pair that corresponds to the number of molecules on the cell, you can skip this step and the next)įigure 1. (Note: if your calibration standards were acquired as one tube, first export the individual peaks, and then re-import the new FCS files into FlowJo). Place your calibration standard samples into their own group. The following steps guide you through creating the standard curve, calculating the line that fits the curve, and ultimately deriving the number of molecules on the surface of a cell in your experiment: Have three or more standards that cover the anticipated range of expression on your target cells, together with a blank. In FlowJo v10, we need to start with data from your calibration standards. The strict measurement being determined here is the molecules of equivalent fluorescence (MESF). Note: In the following example, we assume one bound antibody per molecule, which may not be true depending on antibody class, distance between molecules, and number of targeted epitopes on a given molecule. With the standard curve we derive a linear relationship between fluorescence intensity and number of molecules on a given cell. Considering that fluorescence intensity is correlated with molecules on the surface of the cell, can the relationship between the two be quantified? If so, how can we use that relationship to calculate the number of molecules on the surface of a cell in a given experiment?Īs with all indirect measurements, a standard curve must first be created using calibration standards (for example, cytometric bead arrays), to establish the relationship between the fluorescence intensity measurements and the antibody binding to its target molecule. Measuring the fluorescence intensity of cells and particles is routine and the basis of the vast majority of inquiry in flow cytometry.
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