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Improved Flow Cytometry for More Powerful Cell Analysis

Thu, 02/06/2014 - 1:05pm
Jessica Reed, Product Manager, EMD Millipore, Billercia, Mass.

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Figure 1: Data from the guava easyCyte system with Spherotech 8 Peak rainbow beads. Images: EMD Millipore
 
  

The benefits of flow cytometry are well known. The popular technique allows researchers to explore data on a cell-by-cell basis, as opposed to other analysis methods which only offer population-based or averaged information. In addition, flow cytometry can give users absolute percentages of what each marker or dye is reporting.

Multicolor flow cytometry can dramatically increase the discriminatory power of cell analysis. However, traditional multicolor flow cytometers can be a challenge to use due to the need to manage optics and manual compensation, choose fluorescent dyes and analyze results. The complex data analysis requires deep expertise and large dedication of user resources.

The guava easyCyte flow cytometry systems (EMD Millipore, Billercia, Mass.) offer researchers a simple benchtop method for multicolor flow cytometry. These systems provide single blue (488 nm), dual blue and red (642 nm) or triple blue, red and violet (405 nm) excitation lasers to provide up to 12 simultaneous detection parameters, including 10 fluorescent colors plus forward and side scatter for size and granularity determination. Single-sample and multi-sample processing options are offered, and high-throughput analysis is possible with robotic sample trays that automatically handle 96-well micro­plates and up to 10 sample tubes. The systems are capable of detecting mammalian and micro­bial cells and beads.

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Figure 2: 10-μL adult human blood was stained for 20 min at room temperature with a cocktail containing anti-CD45 PerCP-Cy5.5, anti-CD3 Brilliant Violet 421, anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CD16-PE, CD56-PE, anti-CD19 Brilliant Violet 510, anti-CD45 RA APC and anti-CD62L FITC. After incubation, cells were lysed and fixed with 180-μL Guava lysing solution for 15 min at room temperature. Samples were then acquired on the guava easyCyte 12HT system. Lymphocyte cells (CD45+) were gated into a SSC vs. CD3 plot. T-cells (CD3+ and CD45+) were gated into a plot comparing CD4 and CD8 positive cells. CD4 and CD8 naïve memory cells were further discriminated by evaluating each population in a CD45RA (naïve T-cells) vs CD62L (memory T-cells). To separate the natural killer (NK) and B cells from the lymphocytes, CD3-negative cells were gated into a plot comparing CD19 (B-cells) and CD16+56 (NK cells).
 
  

In all guava easyCyte systems, a sample of fluorescently labeled cells is aspirated into a microcapillary flow cell, which eliminates the need for sheath fluid and provides absolute cell counts. This results in less complexity, smaller samples and minimal waste, saving researchers time and money. In addition, the flow cell is self-aligning and user-replaceable, which reduces downtime and service visits.

Three-laser excitation
After the sample is aspirated into the flow cell, red, blue and violet lasers excite fluorophores, and each cell emits signals that are individually detected by photomultipliers and photodiodes. The addition of the violet laser allows for multiplexed assays that query multiple cell parameters simultaneously, permitting the user to perform complex analysis without the need for compensation.

In two- and three-laser systems, the lasers overlap spatially and are modulated out of phase at high frequency so that each par­ticle is sampled many times as it travels through the overlapped beams. Modulation is particularly important for identifying dyes which have over­lapping emissions, such PE-Cy7 (blue laser excitation) and APC-Cy7 (red laser excitation). Unlike spatially separated beams, modulation eliminates the need for time-delay calibration, sim­plifying the overall operation of the instrument.

Figure 1 shows the sensitivity of the guava easyCyte system, presenting the data obtained from Spherotech 8 peak rainbow beads, a typical flow cytometry control. All eight peaks are clearly resolved in all channels utilizing the three lasers.

Software system
The guava easyCyte system’s software suite provides access to modules for acquisition and analysis, as well as instrument setup and maintenance. The accompanying InCyte software has an intuitive, easy-to-use interface that allows researchers to focus on data at either the sample or experiment level. It simplifies setup and analysis of plots with drag-and-drop features, while automated compensation makes it easy to perform complex, multi-color assays. In addition, its instant update feature responds to changed analysis conditions for real-time viewing.

The software offers an effective way to obtain a macroscopic view of experiments and compare different experiments in real time. It is especially useful for interpreting the results of high-throughput cell-based assays.

Applications
The multicolor flow cytometry systems can be used for a variety of cell analysis applications. For example, multiplexing cell health parameters can give deeper insights into various cell function pathways, including cell death, protein activation and mitochondrial membrane potential loss.

Figure 2 shows how the system can be used for easy identification of subpopulations in a heterogeneous population; in this case, for immunological phenotyping. Adult human blood was stained for 20 min at room temperature with a cocktail containing a set of antibodies often used to identify key lymphocytes, including B, T and NK cells. After incubation, cells were lysed and fixed with 180 µL of Guava lysing solution for 15 min at room temperature. Samples were then acquired; separation was clearly visible, providing the population percentage and counts of all the immune system cells.

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