The emerging field of high-throughput (HT) flow cytometry is extending the capabilities of cell-based screening technologies into the profiling of compound libraries. The screening of molecules for effects in living cell populations has become an integral step in virtually every drug discovery program. High-throughput flow cytometry is a particularly useful approach for screening compounds in cell models where multiple readouts are desired. This can be performed using the Invitrogen Attune NxT Flow Cytometer with Attune NxT Autosampler, where 96- and 384-well plates can be acquired and analyzed. In addition, up to 16 different parameters per cell can be measured on the Attune NxT Flow Cytometer, and thousands to millions of cells can be analyzed in minutes at up to 35,000 cells/sec and 1 mL/min sample input.
The Attune NxT Flow Cytometer uses a unique volumetric sample and sheath fluid delivery system. Samples are introduced to the Attune NxT Flow Cytometer with syringes, producing accurate measurements of the volumes of acquired samples and thus accurate calculation of cell concentrations. Given time savings, high content, and assay robustness, high-throughput flow cytometry provides the capacity needed as part of an effective and practical systems biology approach. These recent developments expand the role of flow cytometry in drug discovery and life science research and are a key factor in understanding cellular and molecular networks. In this application note, consistency across 96-well plates was examined. In brief, lysed human whole blood was labeled with fluorophore-conjugated monoclonal antibodies for CD45, CD3, CD4, and CD8 targets. The labeled cells were fixed, then added in equal amounts to each well of two 96-well plates, and acquired on a 4-laser Attune NxT Flow Cytometer with Autosampler. The concentration statistic representative of events/µL of any given population and percent positive are examined in depth.
The following protocol was used for sample preparation, acquisition, and analysis on the Attune NxT Flow Cytometer. Please see the user guides for detailed instructions on setting up an experiment and running samples [1-3]. Table 1 lists the lasers, detector bandpass filters, and fluorophores used.
Table 1. Instrument configuration and antibody conjugates used.
|Target||Fluorophore||Detector||Laser wavelength (nm)||Bandpass filter (nm)|
|CD45||Alexa Fluor 488 dye||BL1||488||530/30|
|CD8||Pacific Blue dye||VL1||405||440/50|
Table 2. Preparation of FMO controls.
|Sample||Unlabeled cells (from step 1.12)||CD45 Alexa Fluor 488||CD3 R-PE||CD4 APC||CD8 Pacific Blue||Labeled cells (from step 2.11)|
|Unlabeled sample||100 µL||None||None||None||None||None|
|FMO, no CD45 Alexa Fluor 488||100 µL||None||5 µL||5 µL||5 µL||None|
|FMO, no CD3 R-PE||100 µL||5 µL||None||5 µL||5 µL||None|
|FMO, no CD4 APC||100 µL||5 µL||5 µL||None||5 µL||None|
|FMO, no CD8 Pacific Blue||100 µL||5 µL||5 µL||5 µL||None||None|
|4-color labeled sample||None||None||None||None||None||100|
Figure 2. Spillover matrix for the four fluorophores used in this experiment.
Figure 4. Representative data and gating example. Plot A is gated on all events with CD45 vs. SSC showing all three WBC populations, distinct from debris. A gate is drawn around the CD45+ lymphocyte population, used to gate plots C, D, and E. Plot B of the time parameter vs. SSC is gated on all events, demonstrating steady and consistent data acquisition over the entire sample collection. Plot C of CD3 vs. CD4 shows expected patterns and identifies CD45+ CD3+ CD4+ T helper cells with the percent positive of the parent lymphocyte gate displayed in each quadrant. Plot D of CD3 vs. CD8 shows expected patterns and identifies CD45+ CD3+ CD8bright+ T suppressor cells as distinct from CD45+ CD3– CD8dim+ cells, with the percent positive of the parent gate displayed in each quadrant. Plot E shows mutually exclusive T cell populations of CD4 vs. CD8 where no co-positivity is demonstrated, with the percent positive of the parent gate displayed in each quadrant.
Figure 5. Display of the percent positive of three gated cell populations over two 96-well plates. These statistics are displayed for each well of both 96-well plates. Coefficients of variation for each measurement were less than 4%.
Figure 6. Display of the concentration statistic of three gated populations over two 96-well plates. The coefficient of variation is shown for each.
Figure 7. Consistency of all 192 wells for analysis of percent positive of parent gate of the three identified populations.
Figure 8. Consistency of all 192 wells for analysis of concentration of the three identified populations.
The combination of fluorophores and antibodies in this experiment were chosen specifically to reduce the amount of compensation. The four fluorophores are each detected by a different laser. Compensation values were determined using automatic compensation and found to be minimal (Figure 2). The FMO controls were useful to ensure optimal placement of gates. Acquisition of each 96-well plate was completed in approximately 43 min. Consistent results were observed across wells on a 96-well plate and between 96-well plates, with coefficients of variation observed below 4% for each population of cells when analyzing percent parent and concentration in cells/µL.
Figures 5–8 show the consistency across each 96-well plate and for all 192 replicate samples for percent positive and concentration of the three cell populations. The CD45+ lymphocytes are a subset of all events, including all debris events. The CD45+ CD3+ CD4+ and CD45+ CD3+ CD8+ populations are gated on the CD45+ lymphocyte gate, and thus represent T cell subsets.
Another method for evaluating consistency is the Heat Map analysis function in Attune NxT Software. The Heat Map view shows a virtual plate layout that represents the wells available on the 96-well plate. The Heat Map Setup panel allows selection of the statistic, gate, and parameter for visualizing the data from the experiment, and definition of the display mode and transition values to analyze the data at a glance. The Heat Map view may be used for analyzing tube-based data in addition to plate-based data. Figure 9 illustrates this visual display, using the concentration (in cells/µL) of the CD45+ CD3+ CD4+ cell population. The selected parameter value for each well is displayed in the corresponding graphic well, and the color is selected by the location of the value on the color transition display.
Figure 9. Heat Map view and analysis. (A) Heat Map Setup panel and (B) display using settings as defined in (A). The transition values and colors are used to display the results in an easy-to-view format.
Automation helps maximize reproducibility between experiments and reduces inter-assay variability and inter-operator variability. With low CVs across and among plates, the Attune NxT Flow Cytometer with Autosampler offers a reliable and high-throughput approach for multiparametric cell screening from 96- and 384-well plates, allowing analysis from smaller sample quantities to maximize efficient use of limited numbers of cells.
This combination of higher-throughput with higher-content flow cytometric analysis of as many as 16 parameters for each cell, with acquisition speeds of up to 1 mL/min, provides a powerful screening platform for drug discovery and systems biology experiments. For further information, read our application note about recommendations for accurate concentration measurements on the Attune NxT Flow Cytometer .
For Research Use Only. Not for use in diagnostic procedures.