Time: Saving Your Data for Free

Nobody’s got time to think in December and January - Exhibit A: yours truly!

December is a whirlwind of action that we look forward to with mixed parts of dread and anticipation, and then January is one big month of denial as the grant writing season and academic year kicks into gear and we all resist that pressure; hoping to squeeze out a little more relaxation and a few more BBQs with mates.

With all that in mind, let’s keep this brief.

don't waste time (1).png

One of the most underutilised parameters that cytometers can record in your data files is the Time parameter. On most modern cytometers you don’t have to do a thing; the instrument will include it for you by default. Like the title says - it’s free! You don’t have to remember it, or do anything, it will just be there in the background, ready to save your bacon data. If you are using an older cytometer, you may need to select the Time parameter, but it should be as easy as a single button click when you are selecting the rest of your parameters.

Plotting Time against any other parameter will show you the event rate over the duration of the analysis. This can highlight inconsistencies in the flow rate (especially important for QC), moments in the sample where a blockage occurred or where air was aspirated instead of sample. All of these can have a negative impact on your sample, by altering your CV and/or creating a bunch of aberrant events that are not in fact cells.

The example in Figure 1 clearly shows that at the end when the tube was run dry a large collection of non-specific "events" are collected. This is created by a combination of the slurry of cells from the bottom of the tube that surges through in a big clump and light reflected and refracted from air bubbles as they pass through the flow cell. This tube is a FITC compensation control (i.e., contains no fluorochromes except FITC), and yet, what appears to be a FITC and PE double-positive population can be seen.

 Figure 1.

Figure 1.

When a gate is drawn to exclude these "events" (shown in Figure 2) the expected result from the data can be observed (i.e. FITC positive only, this cleans up even more when FSC/SSC gates and viability gates are applied too).

 Figure 2

Figure 2

And just to be contrary, these erratic events that appear when the sample is run dry can be positive AND/OR negative. As shown in Figure 3, where each of the different fluorochromes is plotted against time, take note where the fudgy stuff (yes, that is the real technical term) falls with respect to fluorescent intensity.

 Figure 3.

Figure 3.

In short, a few seconds of your Time is all it takes to check your sample for acquisition errors, and applying a gate to eliminate errors such as blockages, surges (clumps) and the tube running dry could mean the difference between meaningful data and rubbish.