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Flow cytometry is fast becoming a transformative tool in all phases of the drug discovery process. The ability to examine individual cells at the rate of thousands per second has made flow cytometry an attractive technology for investigating new drug candidates, evaluating the effectiveness of cancer treatments, and understanding mechanisms of cell health [1,2]. Continued advances in detection technologies offer enhanced efficiencies in identifying and characterizing potential cancer therapies. With its high sampling rates, fast detection speeds, and clog resistance, the Invitrogen Attune NxT Flow Cytometer enables accurate cell measurements for the evaluation of cancer drugs. And because it allows the simultaneous detection of up to 14 colors within a single sample, the Attune NxT Flow Cytometer is well suited to multiparametric analyses.

Dose-response studies with the Attune NxT Flow Cytometer and Autosampler

Flow cytometry is an effective technique for dose-response studies, providing quantitative data at the individual cell level on a large sample size across a wide range of drug concentrations in a short amount of time. Traditional cytometry has proven to be ineffective in many high-throughput protocols because most flow cytometers rely solely on hydrodynamic focusing technology, which cannot handle large, clumpy cells without clogging the system. These clogs lead to cell loss and machine downtime and require user intervention to resolve issues. However, the fluidic system of the Attune NxT Flow Cytometer was designed to minimize the potential for clogging and sample loss and, because it is syringe pump driven, has an automated function to unclog the flow cell if needed. Moreover, the acoustic-assisted hydrodynamic focusing technology employed by the Attune NxT Flow Cytometer effectively interrogates cells at high sampling rates, even in dilute samples, enabling the use of reduced sample preparation procedures.

The application note “Flow cytometry analysis of dose-response for apoptosis induction” provides an easy and efficient protocol for evaluating therapeutic cancer drugs using the Attune NxT Flow Cytometer together in conjunction with the Attune Autosampler, which allows for rapid processing of multiple samples in 96-well plates. In this protocol, Jurkat cells (human T cell leukemia) are grown and treated in 96-well plates with eight concentrations of four different anti-cancer drugs—staurosporine, cycloheximide, camptothecin, and etoposide (Figures 1 and 2)—and then evaluated for apoptosis using the Invitrogen CellEvent Caspase-3/7 Green Detection Reagent and analyzed on the Attune NxT Flow Cytometer (Figure 1). A total of 20,000 cells are acquired from each well at a speed of 200 μL/min. Additional time savings are obtained by adding the CellEvent Caspase-3/7 Green Detection Reagent directly to live cells in media, eliminating the need to wash, fix, or permeabilize the cells prior to analysis. Also, the Attune NxT software produces a heat map view that provides a quick visual evaluation of the plate results, allowing you to simultaneously visualize multiple variables in the experiment (Figure 2).

cancer-attune-fig1

Figure 1. Dose-response curves for evaluating the effectiveness of four anti-cancer drugs in inducing apoptosis in Jurkat cells. Apoptosis was induced in Jurkat cells (human T cell leukemia) using eight different concentrations of the indicated drugs, and then detected using the Invitrogen CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit. Each drug concentration (shown from high to low on the x-axis) was run in triplicate. Statistics from the whole plate were exported as a single CSV file and analyzed using GraphPad Prism™ statistical software.

cancer-attune-fig2

Figure 2. Results displayed with the Attune NxT software heat map. The Invitrogen Attune NxT Flow Cytometer software produces a heat map view that provides a quick visual evaluation of the plate results. This view uses a range of colors along with a numerical value to indicate the percentage of cells in each well that were labeled with a particular probe, (e.g., apoptotic cells, as indicated by a positive signal from the Invitrogen CellEvent Caspase-3/7 Green Detection Reagent). The percent of positive-stained cells is displayed in each well, and the well color transitions from purple to red as the percentage increases, with red wells containing the highest percentage of apoptotic cells.

Cell health assessment with the Attune NxT Flow Cytometer

Multicolor flow cytometry provides a means of analyzing large numbers of individual cells for multiple cell health parameters in a short amount of time. For example, the Attune NxT Flow Cytometer enables the simultaneous measurement of up to 16 parameters (14 different emission channels plus forward and side scatter) of cell health at the single-cell level, with sampling rates of up to 35,000 cells/sec at 1 mL/min. This high sampling rate allows for thousands to millions of cells to be analyzed in minutes.

The application note entitled “Life, death, and cell proliferation: Multiplex single-cell analysis with minimal compensation” combines proliferation, viability, and apoptosis functional assays (Table 1) in a single experimental design to simultaneously examine a CD4+ subset of stimulated human T lymphocytes. This protocol requires minimal setup. In addition, fluorescence color compensation requirements can be minimized or even eliminated by using the Attune NxT Flow Cytometer equipped with 405 nm violet, 488 nm blue, 561 nm yellow, and 637 nm red lasers, in conjunction with spectrally compatible fluorophores that show minimal spectral overlap.

The Invitrogen LIVE/DEAD Fixable Violet Dead Cell Stain (405 nm violet laser) provides a simple method of distinguishing live and dead cells using an amine-reactive dye that maintains identification of dead cells after fixation. The Invitrogen CellTrace Far Red Cell Proliferation Dye (637 nm red laser) facilitates the identification of each generation of cells based on fluorescence intensity. The combination of the LIVE/DEAD Fixable Violet viability dye, the CellTrace Far Red dye, and the CellEvent Caspase-3/7 Green apoptosis indicator (488 nm blue laser) enables the identification of three separate cell populations—live, proliferating, and apoptotic cells; the PE anti-CD4 antibody conjugate (561 nm yellow laser) was added to these functional probes to identify the CD4+ population during the analysis.

With this experimental design, apoptotic cells can easily be discriminated from live and necrotic cells (Figure 3). Compared with unstimulated cells, stimulated cells have a higher percentage of apoptotic cells, demonstrating the activation of the caspase enzymes and the early stages of apoptosis. Using the PE anti-CD4 antibody conjugate, we found that the majority of the proliferating cells, as identified by the CellTrace Far Red reagent, are CD4+ (Figure 4).

Table 1. Three spectrally compatible cell viability assays for characterizing cell populations by flow cytometry.

Kit Dye Laser,
bandpass filter
Function Utility
CellTrace Far Red Cell Proliferation Kit Cell-permeant amine-reactive dye 637 nm red laser,
670/14 nm BP
Covalently binds to amine groups, allowing the determination of different cell generations based on fluorescence intensity When all three probes are used together, three separate cell populations can be identified—live cells, proliferating cells, and apoptotic cells. For more information, go to thermofisher.com/
flow-cellviability
LIVE/DEAD Fixable Violet Dead Cell Stain Kit Cell-impermeant amine-reactive dye 405 nm violet laser,
440/50 nm BP
Allows identification of dead cells before and after fixation based on membrane integrity
CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit Cell-permeant fluorogenic caspase substrate 488 nm blue laser,
530/30 nm BP
Enables detection of activated caspase-3 and -7 in apoptotic cells
cancer-attune-fig3

Figure 3. Data acquisition on the Attune NxT Flow Cytometer for stimulated human lymphocytes. (A) Gating on density plot of SSC-height (SSC-H) vs. SSC-width (SSC-W) removes cell aggregates from the analysis. The singlet population is displayed in (B) through (E). (B) Histogram of Invitrogen LIVE/DEAD Fixable Violet Dead Cell Stain fluorescence. The large peak on the left is the population of live cells; whereas the smaller peak on the right represents the dead cell population. (C) Histogram of Invitrogen CellEvent Caspase-3/7 Green Detection Reagent fluorescence. The large peak on the right is the apoptotic cell population. (D) Histogram of R-phycoerythrin (PE) anti-CD4 antibody fluorescence showing CD4+ and CD4 cells. (E) Histogram of Invitrogen CellTrace Far Red Cell Proliferation Dye fluorescence, showing the individual generations of proliferating T cells. The analysis of the cell health of these stimulated human lymphocytes continues in Figure 4.

cancer-attune-fig4

Figure 4. Data acquisition on the Attune NxT Flow Cytometer for stimulated human lymphocytes. Cells were first gated on the singlet cell population as shown in Figure 3. (A) Density plot of singlet population showing the discrimination of live and dead cells using Invitrogen LIVE/DEAD Fixable Violet Dead Cell Stain fluorescence. A gate was created around the live cells, which represent 67.7% of the singlet population. (B) Density plot of Invitrogen CellEvent Caspase-3/7 Green Detection Reagent fluorescence vs. Invitrogen CellTrace Far Red Cell Proliferation Dye fluorescence. Fewer apoptotic cells are seen in early generations (upper right), whereas far more apoptotic cells are seen in later generations (upper left). (C) Density plot of CellEvent Caspase-3/7 Green fluorescence vs. LIVE/DEAD Fixable Violet fluorescence. Live cells (26.5%), dead cells (34.6%), and apoptotic cells (38.3%) can be identified. The live cells identified in (A) were further analyzed: (D) Gated on the live cells from (A), a histogram of live singlet cells labeled with CellTrace Far Red dye, showing multiple peaks representing each generation of this proliferating population. (E) Gated on the live cells from (A), a density plot of PE anti-CD4 antibody fluorescence (labeling T helper lymphocytes) vs. CellTrace Far Red fluorescence, showing the increasing generational proliferation of CD4+ cells.

Streamline workflows with the Attune NxT Flow Cytometer

The Attune NxT Flow Cytometer was designed to facilitate high-throughput screening assays in drug discovery applications. The Attune NxT acoustic focusing technology simplifies sample preparation requirements, provides rapid throughput rates that are up to 10 times faster than traditional flow cytometers, and offers the Attune Autosampler option to accommodate 96-well (and 384-well) plates. The increased data fidelity, faster acquisition rates, and improved fluidics expand the scope and power of flow cytometry into areas such as cancer, synthetic biology, and drug discovery. Together with our wide selection of flow cytometry antibodies and reagents, the Attune NxT Flow Cytometer and Autosampler streamline high-throughput workflows, saving time and expanding resources. Visit thermofisher.com/flowappnotesbp75 to download these application notes.

References

  1. Sharma P, Muthuirulan P (20 September 2016). Flow cytometry: breaking bottlenecks in drug discovery and development. Drug Target Review.
  2. Thayer M, Draper D, Saims D et al. (2016) In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16–20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 76(14 Suppl): Abstract 3242. doi: 10.1158/1538- 7445.AM2016-3242.

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