The Basics of High-Throughput Screening (HTS), through Flow Cytometry
Scientists face an overwhelming array of techniques to integrate and investigate cellular features. Traditionally, many of these methods have been slow and labor-intensive. The future demands rapid solutions for quicker patient diagnoses, earlier project completions, extensive library screenings, accelerated cures, and faster patent processes. Flow cytometry emerges as a promising solution. Flow cytometry is the gold standard for antibody and phenotypic screening, offering unmatched multiplexing, flexibility, speed, and reproducibility.
Why Flow Cytometry?
Flow cytometry is a powerful technique for measuring physical and biochemical characteristics of cells using fluorescence. It offers:
- Rapid throughput: Up to tens of thousands of particles per second.
- Single-cell or bead analysis: Measures individual cells or beads.
- Multiparametric measurements: Simultaneous analysis of multiple parameters from one cell.
- Data rich: Collect information on millions of cells.
What is Flow Cytometry?
Flow cytometry is a well-established, widely used, and powerful technique for measuring intrinsic and extrinsic, physical and biochemical characteristics of cells and cell-like particles using fluorescence. It rapidly counts and measures analytes on, in, or produced by individual cells, allowing for diverse biological trait and indicator measurements.
Flow Cytometry Basics:
- Light Scattering & Fluorescence: Measures properties of cells.
- Instrumentation: Cells in liquid are moved single file through a tube, detected by lasers and detectors.
- Data Analysis: Signals are converted to digital values and displayed on a computer.
Flow Cytometry Theory: Biological cells or particles have the ability to scatter light and can have intrinsic fluoresce. Flow cytometry exploits these properties for qualitative and quantitative measurement. Use of fluorescently labelled markers such as antibodies, can be utilized to identify a multitude of cellular features on a single cell. Fluorescence is the ability of an entity to be excited by light of a certain wavelength and then emit light of a higher wavelength.
Basic Instrumentation: Cells suspended in a liquid are moved single file through a narrow tube by sheath fluid (flow cell). Lasers and detectors detect light emitted or scattered by the cells. This light is recorded electronically, converted to digital values, and displayed on a computer.
Image: Description of a basic flow cytometer
The flow cell: Hydrodynamic, acoustic or a combination of focusing aligns biological entities in single file using laminar flow. The sample speed is controlled by adjusting sheath fluid pressure, with low speeds optimizing orientation and stability. Stability is crucial, as poor sample quality can disrupt the system. When a biological entity encounters the laser, light scatters. Forward scatter (FSC) detectors measure light influenced by particle size and membrane integrity, distinguishing live cells from debris. Side scatter (SSC) detectors measure light at a 90º angle, providing insights into cell complexity and granularity. FSC and SSC are unique to each entity, helping identify populations in samples. Fluorescence emissions are also measured by detectors.
Image: Depiction of flow cytometer light scatter
Detection channels: Traditional flow cytometers will be fitted with one of more lasers for fluorochrome excitation and various fluorescence detectors to capture light emission unique to that fluorochrome. The number of fluorochromes that can be measured by a traditional cytometer matches the number of fluorescence excitation and detector channels it possesses.
Image: Design of flow cytometer light detection
Parameters: Detector measurement, like forward scatter, side scatter, or fluorescence, is a "parameter," and each detectable entity with its parameter value is an "event." Fluorescence and scatter signals generate electrical pulses, capturing attributes like pulse height, area, and width. To exclude unwanted debris, triggers and thresholds are set at the start of a run. Gain (voltage) is applied to resolve entities from noise, affecting signal range. Initial runs adjust gain for each detector. Negative controls, such as unlabeled cells, should always be included. Data files, called FCS files, store parameter intensities and instrument settings, and can be visualized in formats like histograms and plots.
Fluorochromes: Selecting the right laser excitation wavelength and optimal spectral emission for each fluorochrome is crucial for obtaining a strong detector signal. Online tools like 'spectra viewers' or 'panel viewers' can help visualize the excitation and emission peaks of fluorochromes.
Image: A typical fluorochrome spectra as shown in a viewer
Log amplification in fluorescence studies expands weak signals and compresses strong ones, making it easier to display a wide range of intensities. Linear scaling is better for low dynamic range signals, like in cell cycle analysis. Many tools are available such as fluorescent dyes, antibodies, and proteins label molecules for various analyses, including cell sorting and apoptosis. Some dyes enter live cells, while others only enter compromised ones, distinguishing live from dead cells.
Tandem fluorophores increase color analysis by transferring energy between bonded fluorochromes. Fluorescent proteins indicate protein expression, and quantum dots offer narrower emission peaks. Multi-color fluorescence allows analysis of complex populations, with the number of fluorochromes depending on lasers and detectors.
Fluorescence from labeled entities is focused on by establishing a background detection level. Overlapping spectra can reduce sensitivity, but additional lasers enhance signal definition. Compensation corrects for fluorescence spillover using controls to subtract unwanted signals. Optimizing dye staining is crucial, involving factors like incubation time and dye-to-entity ratio. Proper resolution requires sufficient dye, achieved through titration to maximize the signal-to-noise ratio.
Sample preparation: It is important that the sample is presented as a single cell suspension. This can be achieved by mechanical dissociation and detachment, using enzymatic solutions or calcium chelation, are common for single-cell suspensions. Antibody dilution and buffer washing prevent nonspecific binding. Sample suspensions are incubated with components like primary and secondary antibodies, streptavidin, and fluorochromes. Blank and control samples, with known fluorochrome quantities, are used alongside test samples to optimize flow rates and voltages.
Beads: Flow cytometry beads are non-biological microspheres used to enhance the process through quality control, standardization, and compensation. They reduce variation in instrument alignment, improving data accuracy, reliability, and reproducibility.
- Quality control beads are used for calibration, compensation, and counting, ensuring instruments meet specifications.
- Compensation beads minimize fluorescence spillover in multicolor panels, especially when auto compensation isn't available.
- Counting beads helps calculate particle concentrations by comparing bead and cell counts, providing accurate cell numbers. They are added after lysing, staining, and washing. An exact volume of beads is mixed with a known cell sample, and the ratio of events is used to determine cell numbers.
Data Analysis: Flow cytometry uses detector signals to create pulse peaks for SSC, fluorescence, or FSC, which are visualized in “dot plots”. Gating is a key tool for isolating cellular subsets and eliminating unwanted events like dead cells and debris. Proper gating enhances data accuracy.
- Software is key in translating the electrical signals into visualizations that help interpretation like dot plots, histograms, heatmaps and cluster diagrams. ‘Dot plots’, are where the peak areas or peak heights are graphically visualized for biological entities passing through the flow path. Each dot represents one event from the flow cells enabling the visualization of millions of entities on one graph.
- Gating: The traditional path of flow cytometric analysis is through “gating.” Gates and regions are placed around populations of cells with common characteristics, usually forward scatter, side scatter and marker expression, to investigate and to quantify these populations of interest.
- Singlets: If two biological entities stick together, or pass through the flow cell too closely, both the peak area and width became larger compared to those of single cells. A singlets gate can be used to eliminate biological entities that show an increase in peak area without an increase in peak height. It important to gate doublet events out as they can cause artificially high fluorescence results for a given entity.
High-Throughput Screening (HTS)
High-throughput screening (HTS), uses minimal assay volumes, which lowers the cost and is ideal for screening vast numbers of sample usually in 96- or 384-well microplates. HTS screening typically has less parameters to investigate but a significantly larger sample size.
Automation is beneficial ranging from:
- Full integrated automation systems consisting of robotic arms for transporting assay-microplates for sample and reagent addition, mixing, incubation, and detection.
- Or simpler plate loading instruments to support unsupervised assay analysis.
iQue® HTS by Cytometry
The iQue® HTS Cytometer provides the perfect combination of multiparametric cell and bead analysis with the speed and usability suitable for HTS supported by powerful Integrated iQue Forecyt® software. The key differentiator of iQue® Platform is in the air-gap technology that allows continual collection and analysis samples in as rapid as 5 minutes for a full 96-well plate.
Simple to use, flexible and the faster alternative.
- iQue® HTS by Cytometry rapidly measures and screens multiple parameters including cell viability, immunophenotype, and cytokine secretion in a single well, with the ability to screen millions of cells.
- Sample carryover is avoided by generating air gaps between samples and by automatically washing the probe between samples.
- Easily integrated with multiple plate loader robotic systems to expand screening capacity.
- User friendly iQue Forecyt® software with additional availability of 21 CFR Part 11 compliance software.
HTS by Cytometry Resources
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FAQ
Within drug discovery, chemistry, biology and materials science, this is a process where large numbers of moieties or samples are simultaneously examined by automation, detectors and computing, to identify samples or complexes of desired properties. Hence a scientist is able to quickly conduct tens of thousands of pharmacological, chemical, antibody, genetic, substance or other assays.
Depending on the results of initial assays, a scientist can perform follow up assays by further clarifying and investigating in detail, any initial sample or moiety of interest hits.
The is an assumption that a sample or moiety, contained within any given sampling or moiety holding vessel, will not interact with another sample or moiety within the same holding vessel.
High-throughput screening techniques include high-throughput screening by cytometry, genome sequencing, coulter counters, time-of-flight mass cytometry, imaging mass cytometry, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, mass spectrometry and chromatography.
High-throughput screening (HTS) by cytometry mainly relies of the detection of fluorescent signals, and permits rapid analysis of large libraries of moieties or cells, to assess effects on specific biological targets. HTS by cytometry brings together the high-resolution capabilities of flow cytometry and the power of HTS. This enables simultaneous multi-parameter analysis, making it ideal for assessing cell populations and for screening compound libraries, drug discovery, biomarker discovery, and cell-based assays.
HTS by cytometry can use minimal samples and assay volumes. After initial screening, samples may be subjected to further detailed examination, using slower traditional flow cytometry or other techniques.
HTS heavily relies on automation using robotic liquid handling devices, microtiter well plates and robust data processing software to streamline the testing process.
Ultra-low adherence cell repellent microtiter plate surfaces, facilitate the rapid development of HTS assays in 3D tissues such as organoids and spheroids.
HTS can be fast, flexible, scalable, efficient and cost effective.
At present we can’t rely on AI models to fully predict biological/chemical/material compound interaction. Hence actual laboratory work with real-life samples is required. AI may be used, with caution, in assessing final HTS data.