The Impact and Exclusion Methods of Cell Aggregates

Cell aggregates refer to the phenomenon where two or more cells adhere to each other (usually two cells).

Ideally, cells pass through the laser irradiation area one by one through flow cytometry. The fluorescence signal generated by cells passing through the laser irradiation area is received by the PMT and converted into an electrical pulse signal, as shown in the figure:

From entering the laser irradiation area to leaving, the electrical pulse signal goes from 0 to peak and then back to 0, which is recorded as one peak. H is the pulse height, representing the peak value of the pulse signal, W is the pulse width, referring to the time when cells pass through the laser irradiation area, and A is the area of the pulse signal.

In reality, due to various reasons, cells may stick together and pass through the laser irradiation area continuously, which is recognized as one signal by the instrument.

When two cells are squeezed together and pass through the instrument, compared with a single cell, the measured height remains unchanged, while the width and area become twice as large. This phenomenon is called an increase in FSC-Area and Width while FSC-Height remains unchanged, as shown in the figure:

When analyzing data, if cell aggregates are not excluded, they may cause misleading results. For example:

Suppose that some cells in the sample carry GFP green fluorescence, while others do not. To select cells with GFP signals, there may be same situations for cells in the buffer:

A cell aggregate composed of one cell with GFP and one cell without GFP (red box) is assumed to be a cell with GFP if not excluded, and will be collected by flow cytometry. Therefore, when analyzing cells with GFP, cells without GFP will be mixed into the data, resulting in a higher proportion of cells with GFP than the actual proportion. If selecting cells with GFP, cells without GFP will be mixed in, affecting the purity of the collected cells.

Since cell aggregates can have such a significant impact on results, what are the methods to exclude them during cell analysis?

01 FSC-A and FSC-H

Generally, we use FSC-A and FSC-H to exclude cell aggregates. As mentioned earlier, when normal single cells are irradiated by the laser in the flow cytometer, both H and A increase proportionally. However, when cell aggregates pass  through, H remains unchanged while A increases. Therefore, cell aggregates can be perfectly excluded by using the FSC-A (Area) and FSC-H (Height) parameters of the cells.

02 FSC-H and W

Since the pulse width of a doublet is larger than that of a single cell pulse, it is basically twice the width, so we can also use FSC-H and W to exclude cell aggregates. Because H is independent of W, this method is not affected by area and is highly recommended. However, it should be noted that this analysis method may not be suitable if there is a large difference in the size of cells in the sample being analyzed.

Using the above methods can perfectly exclude cell aggregates and avoid false positive effects caused by cell aggregates. However, if there are too many cell aggregates, even if they are excluded during data analysis, they still have a significant impact on experimental results and instrument pipelines. Therefore, preventing cell aggregates during sample preparation is the preferred solution.

During sample preparation, attention should be paid to the following points to reduce the occurrence of cell aggregates:

· Thorough digestion and mixing;

· Prepare the sample as fresh as possible, especially for single-cell suspensions prepared from tissue dissociation;

· Filter through a 200-mesh sieve before running on the instrument;

· Mix thoroughly by blowing before running, and avoid long periods of standing.

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