This online tool guides you through flow cytometry panel design, providing a simplified, customizable experience to fit your flow cytometry panel design needs. Our Invitrogen Flow Cytometry Panel Builder can help you choose fluorescent antibody conjugates for your flow cytometry panel. You can find the Fluorescence SpectraViewer at /spectraviewer. An additional feature of the SpectraViewer is the spillover table function, which shows fluorescence overlap (or spillover) for each dye in each channel ( Table 2). Figure 5 shows an example of a five-color panel. You can enter your instrument laser and filter configuration ( Figure 4), then select the fluorophores under consideration. The Molecular Probes Fluorescence SpectraViewer is an online tool that displays the excitation and emission spectra for fluorescent dyes and proteins, facilitating selection of appropriate dyes for your multicolor experiment. Online tools: Fluorescence SpectraViewer and Flow Cytometry Panel Builder Staining Index was determined on the BD™ LSR II Flow Cytometer with FACSDiva™ version 6.1 software. †BP Em filter = bandpass emission filter, in nm. *Approximate fluorescence excitation (Ex) and emission (Em) maxima for conjugates, in nm. The SI can be useful for comparing histograms of cell populations stained with different fluorescent conjugates of the same antibody ( Table 1, Figure 3).įluorophore component of conjugate (Conjugate Cat. , the Stain Index (SI) takes these two parameters into account ( Figure 2). However, the relative brightness of a fluorophore-conjugated antibody is determined not only by the intensity difference between stained and unstained cells, but also by the intensity distribution spread of the unstained cell population. To calculate a simple S/N, divide the median fluorescence intensity (MFI) of the positive cells by that of the negative cells ( Figure 2). The signal-to-noise ratio (S/N) is one measure of the sensitivity of an assay and its ability to detect differences between stained and unstained populations. Background fluorescence- e.g., from nonspecific staining, cellular autofluorescence, and instrument noise-can affect the ability to resolve the fluorescence of the antibody conjugate–stained cell population (positive) from that of the unstained cell population (negative). In flow cytometry, fluorophore brightness is a function not only of the quantum yield and extinction coefficient of the fluorophore itself, but also of the effects of background contributions. Include a cell viability dye in the panel to exclude dead cells and debris from the data.Use fluorophores that are spectrally similar for different cell subpopulations that will be gated and analyzed separately.Use bright fluorophore labels on antibodies for low-abundance antigens and dim fluorophore labels on antibodies for highly expressed antigens.Titrate and optimize each antibody building the right panel is an iterative process.Use a tool like the Molecular Probes Fluorescence SpectraViewer to visualize the spectral overlap of fluorophores.Know the configuration of the instrument being used (laser and filters) before you begin.When designing a multicolor flow cytometry panel, there are several key points to consider: Additional publications on this topic are available. There are some excellent resources available for the beginner, including the Molecular Probes flow cytometry webinar “Multicolor Flow Cytometry Panel Design” by Dr. One of the biggest challenges in multiparameter flow cytometry is selecting the right combination of fluorophores and antibody conjugates so that the need for compensation and spillover adjustments is kept to a minimum while the quality and accuracy of the data are not compromised. Increasing the number of colors and antigens detected, however, increases the complexity of the experimental design, requiring significantly higher attention to optimization, controls, and other details. Additionally, multiparameter experiments can improve efficiency by requiring fewer samples and smaller sample volumes and by increasing sample throughput. The advantages of multiparameter flow cytometry include the ability to perform single-cell interrogation with multiple markers, to correlate cell data using multiple analytes, and ultimately to more accurately define cell populations ( Figure 1). (See a list of the products featured in this article)Īdvances in both flow cytometry reagents and instrumentation allow researchers to run increasingly complex multicolor experiments.
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