Example 1-1A group of scientists (Negron et al., 2020) is studying the primary biological aerosol particles (PBAP) in the atmosphere and its influence on human health. The PBAP can be divided into these groups: high nucleic acid-content particles (HNA), low nucleic acid-content particles (LNA) and pollen. Samples are harvested and stained by SYTO-13 nucleic acid probe. 15 µm polystyrene beads were used in the test to set up threshold, in order to identify the correct group of particles. BD Accuri C6 flow cytometer (BD Bioscience Inc.) was used for flow cytometry.
On the FCS plot above, by taking the beads size and relative fluorescent intensity as reference, we can easily locate every population. Pollen are large size particle (15μm to 200μm) and rich in nucleic acid; HNA and LNA are similar in size but HNA has much more nucleic acid. In any further FCS test, the relative location of each population shall not show significant change otherwise it means that the sample encountered some problem. Please notice: unless mentioned in the manual of the beads, the absolute size of the sample cannot be measured by comparing with beads. Source: Negron, A., DeLeon-Rodriguez, N., Waters, S., Ziemba, L., Anderson, B., Bergin, M., Konstantinidis, K. and Nenes, A., 2020. Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia. Atmospheric Chemistry and Physics, 20(3), pp.1817-1838. |
Example 1-2Another example is from PBMCs cell grouping. Isolating PBMCs is simple for experienced labs. However, for researchers who occasionally study PBMCs, the isolation process can drive them crazy. Your sample may have passed many hands before you; you are using some reagent which the functionality is unsure; or simply you are not sure how to gate the right population on the plot. It is time to use the beads to guide you through!
CountBright™ Plus Absolute Counting Beads is from Thermofisher (Catalog number: C36995). It comes with 2 types of size, either 4µm or 7µm. They both have broad range of excitation and emission, which makes them compatible with variable reagent. From the CD19 to CD3 plot, we see that each population has its relative location determined according to the beads (which is located in the right up corner). Among several routine tests, the relative location shall not change, unless any experiment condition change or some problem occurs to the sample. Refer to the instruction of CountBright™ Plus Absolute Counting Beads (Catalog number: C36995), it is possible to achieve absolute cell counting. We may also calculate the cell counts of each group and take the data as reference for further studies. |
Example 1-3Similar to example 2, another product LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit (Catalog number: L34856) is used for bacteria assay in a Flow Cytometer. Bacteria is much smaller compared to cells, on flow cytometer it may be difficult to distinguish them from the debris. The beads here again act as a marker for you to find the desired population (see results in the figure above).
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Example 1-4When transplanting stem cells, the dose is a decisive factor. The current protocol is to use flow cytometry to determine the required dose. The image above shows fluorescence scatter diagram for the identification of the CD34-positive stem cells, of the white blood cells (leukocytes) and of the calibration particles that have been added to the sample. By adding beads in the sample, it is easy to identify the correct population of cells.
J. Neukammer, M. Kammel, J. Höckner, A. Kummrow, A. Ruf: Referenzverfahren zur Messung von Stammzellkonzentrationen. BIOspektrum 21, 294 (2015) |
Example 2-1Hereunder, the image on the left is showing a good liquid flow system; while the image on the right is showing either some cell clog in the fluidic system, or flow failure. In this case, no researcher will be happy to use their precious sample for the test.
Let the beads do the checkup work! You may inject beads sample and check the stability of the run. Plot a time vs a scatter plot to see how even the flow was during the run. If the plot is showing the image on the right, a cleaning may be needed or you have to contact the aftersales service.
(Source: Cheeky Scientist LLC.) |
Example 2-2The change of emission light power may occur with different reasons. On some models of the flow cytometer, the changeable emission power is part of its function; or sometimes simply the laser system problem causes the change of emission power. The unexpected change of emission power is hard to be discovered by running cell sample, and the beads has to be run to test the system.
Rainbow beads from Spherotech (author did not mention the catalog number) injected into a cytometer with different emission power (author did not mention the model of the machine). Emission power is 3.75mW, 7.5mW, 15mW and 30mW separately. The most left peak (the dimmest population) is a group of unstained, autofluorescent beads.
The result reveals that when the emission power going lower, the peaks which indicate lower fluorescent intensity starting to overlap with each other. When the laser power drops to 3.75mW, peak 1, 2 and 3 merged into a plateau and can never be identified. It forms a critical situation in cell research, as the cell samples vary and it often happens that the stained sample has low fluorescent intensity. In order to avoid any unexpected result, you may run the beads ahead of your sample to check the machine. Source: S Tanqri, H Vall, D Kaplan, etc. Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS – Part III – Analytical Issues. Cytometry Part B (Clinical Cytometry) 84B:291–308 (2013) |