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1、Selecting Reagents for Multicolor Flow Cytometry From HotLines Platinum Edition, Fall 2006 By Holden Maecker and Joe TrotterThe availability of flow cytometers capable of detecting 6, 8, or more colors has spurred the discovery of new fluorochromes and the development of antibody conjugates. However
2、, the complexities of choosing antibody combinations are such that simply using a random combination of fluorochromes for a particular set of antibody specificities is unlikely to provide optimal results. On the other hand, with a bit of forethought, one can usually avoid many rounds of trial and er
3、ror. The goal of this article is to provide some simple guidelines to aid in the selection of reagent panels for multicolor flow cytometry, which should result in more successes than failures.The basics: Know your instrumentReagent selection starts with your instrument configuration. The lasers and
4、detectors that you have available dictate whether or not you can excite a given fluorochrome, and whether or not you have enough detectors to read out a given combination of fluorochromes. The design of the optical system also impacts the efficiency with which particular dyes are detected, as do the
5、 instrument settings, including PMT voltages (see the BD Application Note, Establishing Optimum Baseline PMT Gains to Maximize Resolution on BD Biosciences Digital Flow Cytometers at Finally, the choice of optical filters that are used with each detector greatly influences the brightness of one flu
6、orochrome versus another. One can think of filter selection as a give-and-take process: using a wider bandpass filter can increase the ability to detect a given fluorochrome, but may also increase the amount of spillover detected in that detector from other neighboring fluorochromes. A good way to v
7、isualize these effects is by virtual testing of filter combinations using a web tool such as the fluorescence spectrum viewer at Here you can get an idea of predicted spillovers* for particular fluorochromes and filter combinations.Fluorochromes: Go for the brightGiven the many differences in instr
8、ument configuration, it is impossible to universally state the “best” fluorochromes to use in combinations of 6, 8, or more colors. However, if one considers a particular cytometer such as the BD LSR II instrument, it is possible to rank available dyes according to their brightness on that instrumen
9、t (when configured with a specified set of lasers and filters). But how exactly do we define and measure brightness? A good definition of brightness should probably start with resolution sensitivity, the ability to discriminate a dim positive signal from background. Background in a particular detect
10、or is influenced by electronic noise, cell autofluorescence, and spillover from other detectors. To the extent that these factors introduce noise, they also increase the width (or standard deviation, SD) of a negative fluorescence peak (see Figure 1). As such, a good measure of resolution sensitivit
11、y is the stain index,1 defined as D/W, where D is the difference between means of a positive and negative peak, and W is equal to 2 SD of the negative peak.Stain Index = D/WW1W2DFigure 1. Resolution sensitivity (the ability to resolve a dim positive signal from back- ground) depends upon the differe
12、nce between positive and background peak means (D) and the spread of the background peak (W). The stain index is a metric that captures both of these factors.* The predicted spillovers are normalized percentages of omitted fluorescence, and may not necessarily correspond to the percentage used for c
13、ompensation due to the effects of varying detector gains.US Orders: 877.232.8995 Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Prices are subject to change without notice. All applications are either tested in
14、-house or reported in the literature. See Technical Data Sheets for details. Table 1. Stain index of various fluorochrome conjugates on a BD LSR II flow cytometer.ReagentCloneFilterStain IndexPERPA-T4585/40356.3Alexa Fluor 6471RPA-T4660/20313.1APC1RPA-T4660/20279.2PE-Cy7RPA-T4780/60278.5PE-Cy52RPA-T
15、4695/40222.1PerCP-Cy5.52Leu-3a695/4092.7PE-Alexa Fluor 610RPA-T4610/2080.4Alexa Fluor 4883RPA-T4530/3075.4FITC3RPA-T4530/3068.9PerCP2Leu-3a695/4064.4APC-Cy7RPA-T4780/6042.2Alexa Fluor 700RPA-T4720/4539.9Pacific BlueRPA-T4440/4022.5AmCyanRPA-T4525/5020.21,2,3 Fluorochromes listed with the same supers
16、cript number are read in the same detector, and thus would not normally be used in combination.Table 2. Common choices for 6, 8, and 10-color experiments.6-color8-color10-colorFITC or Alexa Fluor 488FITC or Alexa Fluor 488FITC or Alexa Fluor 488PEPEPEPE-Texas Red or PE-Alexa Fluor 610PerCP-Cy5.5PerCP-Cy5.5PerCP-Cy5.5PE-Cy7PE-Cy7PE
