No Compensation Panels
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- Compensation
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Overview of Human and Mouse No Compensation Panels
Designing effective flow cytometry panels can be challenging, especially when it comes to managing spectral spillover and applying accurate compensation. To simplify this process, we have developed a series of five-color no compensation panels, which include a viability dye for both human and mouse samples.
These panels incorporate widely used surface markers for blood and tissue analysis that allow you to identify many populations, such as T cells, B cells, NK cells, and myeloid populations, with no compensation required.
Download a handy overview for easy reference of human and mouse panels. For more detailed panel information, click the links below.
Compensation
When performing multicolor flow cytometry there is a risk of spectral overlap between fluorophores. FITC, for example, has maximal emission at 525 nm, although it actually emits some photons with higher wavelengths which will pass through other filters and end up being detected by unintended PMTs (photomultiplier tubes; detectors). This is known as spectral overlap. So if FITC (with a filter of 525/35) and PE (with a filter of 585/40 in front of its PMT) are combined in a panel, using a 488 nm laser, the relative contribution of FITC and PE fluorophores in both the FITC and the PE detectors must be determined and then removed. This procedure is called compensation (Figure 1) and should be performed for all the fluorophores you are using in a panel.
Fig. 1. Fluorescence compensation. Emission spectra of FITC and PE are shown with a graphical representation of two commonly used filters, 525/50 and 585/40, to detect these fluorophores. Shown in red is the portion of FITC that will be detected in the PE detector and must be subtracted from the PE signal using compensation.
An example of the level of spectral overlap of FITC into the PE and the PE-Cy5 detectors are shown in the flow plots below. The amount of compensation to be applied to PE will be greater than that which needs to be applied to PE-Cy5.
Fig. 2. Relative amounts of FITC fluorescence spillover. Human peripheral blood lymphocytes were stained with CD4 FITC (MCA1267F), and the amount of spillover into filters commonly used to detect PE (A) and PE-Cy5 (B) is shown. FITC gives a greater signal in the filter used to detect PE than the filter used to detect PE‑Cy5. The dotted line represents where the negative population would fall below.
Compensation is a mathematical method developed to account for this spectral overlap and measures the photons deriving from one fluorophore into multiple detectors, so-called false-positive signals. The level of false-positive signals can be determined by single staining your samples with each fluorophore being used. These single stained controls will reveal the level of spectral overlap in each detector. When performing compensation, the fluorophore in the control needs to be identical to the fluorophore used in the sample and its brightness should be at least of a similar level. For more information on controls, go to our dedicated controls in flow cytometry webpage. Figures 3 and 4 show how compensation can be applied to samples to reveal correct levels of staining.
Fig. 3. Analysis without compensation. Human peripheral blood lymphocytes were singly stained with CD4 FITC (MCA1267F) (A) or CD19 PE (MCA1940PE) (B), and both CD4 FITC and CD19 PE (C). In panel A, when compensation was not applied, there is fluorescence spillover into the PE filter (488 nm: 593/52) from FITC (marked by the red box). In panel B, there is a slight amount of spillover from PE into the FITC filter (488 nm: 525/35). Panel C shows how this affects dual staining. Double-positive cells are detected (marked by the red box) even though the markers are mutually exclusive.
Stained cells analyzed with compensation
Fig. 4. Analysis with compensation. When compensation is applied to both FITC and PE channels, we can clearly identify the CD19- and CD4-positive populations with no double-positive cells observed (C).
In addition to fluorescence spillover from one laser there is also the added problem of cross-laser excitation. This is where a fluorophore is also excited by an additional laser than the intended one. It is very important for the fluorescence from single color controls to be measured in all the channels you intend to use. An example of this is shown in Figure 5 where lymphocytes were stained with CD4 PE-Alexa Fluor 750 and acquired on the ZE5 Cell Analyzer with every detector activated. The spectra viewer (Figure 5) shows the predicted cross laser excitation on the left, with the actual data shown by dot plots on the right. In addition to the intended fluorescence from Alexa Fluor 750 detected at 775 nm on the 561 nm laser, fluorescence is also detected at 775 nm from the 355 nm, 405 nm, 488 nm, and 640 nm lasers. Furthermore, due to this fluorophore being a PE-based tandem, there is fluorescence at 578 nm from the 561 nm and 488 nm lasers.
Fig. 5. Spectraviewer image of PE-Alexa Fluor 750 from the ZE5 Cell Analyzer (left) with the corresponding uncompensated two-color dot plots (right). Human peripheral blood lymphocytes were stained with CD3 PE-Alexa Fluor 750 (MCA463P750). Specific staining and fluorescence can be seen at 750 nm from the 561 nm laser (red box). Incomplete energy transfer (FRET) from PE to Alexa Fluor 750 results in a signal from PE at 578 nm from the 488 nm and 561 nm lasers. There is also cross-laser excitation of PE-Alexa Fluor 750, which demonstrates why compensation is required.
Further Reading
More information on fluorophores, fluorescence, and compensation can be found in our flow guide, flow cytometry university, and by watching our webinars.
To determine specific binding you may want to include isotype controls. When performing multicolor flow cytometry, there are also several other important considerations. Careful sample preparation, Fc blocking, inclusion of a live/dead dye, doublet exclusion, and using other appropriate controls are all important. For more information, watch our flow cytometry webinar Optimize Your Flow Cytometry or visit our application resources page.
