Biomedical Engineering Reference
In-Depth Information
of an amine-reactive fluorescent dye. The cells are labeled for a short period of time
and then washed. The dye is covalently bound to the cells, and the cells that received
the most dye have the highest fluorescent signal, whereas those that received less dye
have less fluorescent signal. These four wells are then combined into one well and
stained together. When run on the flow cytometer, one can deconvolute, or separate,
the combined sample back into the constituent wells based on their fluorescent signal
in the barcoding channel.
Of course, because the flow cytometer has many parameters available (many of
which are not used in every experiment on modern instruments with 10-15 para-
meters), more than one fluorescent dye may be used for barcoding purposes. The dyes
can be added in different ratios to create unique fluorescent signatures for each
sample/well; combining two dyes at four concentrations yields 16 (4
4) unique
signatures. Combining three colors yields 64 (4
4) signatures, and so on. We
have been able to combine an entire 96-well plate into 1 tube by using a 4
4
6
matrix of dyes [10]. It is possible to barcode up to 10 different samples using a single
fluorescent dye; however, encoding 4-6 channels is most robust.
FCB can be used in nearly all flow cytometry cell-based assays. Cells can be
barcoded when they are viable or once they have been fixed and/or permeabilized.
Higher signal intensities are obtained, and therefore lower amounts of dye are needed,
in permeabilized cells, but the method works in live cells as well.
4
15.4.1.2 Benefits of Applying FCB to Flow Cytometry
Reduced Antibody Consumption As mentioned above, a major driving force for
developing FCB was to enable us to perform larger screens using minimal antibody
reagent. In the example of a 96-well plate being combined into 1-well using FCB, it is
possible to reduce antibody consumption nearly 100-fold. That is, instead of staining
96 individual samples, only one sample is stained. Obviously, if the barcoding was
expanded to four dyes, one couldmultiplex an entire 384-well plate. This could reduce
the cost per well down to a few pennies, making high-content antibody-based screens
viable on 10,000 or more compounds.
It should be noted, however, that FCB cannot reduce antibody consumption in all
cases. The method relies on the fact that most experiments require only
1000-100,000 cells per sample to be acquired on the flow cytometer. When
10-100 samples are combined, one can still stain a reasonable cell number (about
10,000-10 million cells) in a single well. However, when studying extremely rare
cell types, it may be necessary to acquire data on two million cells. In this case,
because the mass of antibody may be limiting if more than 10 million cells
are stained in one tube (and require more antibody to be added), the benefits of
FCB are slightly reduced. Even in this “worst case” scenario, reagent consumption
is cut two to five-fold. Also, these types of analyses are typically limited to basic
research applications and not drug screening efforts.
Improved Assay Performance When designing assays for cell-based analysis, there
are many experimental components that can lead to variability in data, including
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