Synthetic Cells for Calibration and Standardization

Flow cytometry is a widely used cell analysis platform for characterizing cell populations based on a diversity of physical properties and markers of interest. Its applications range from profiling the activities of immune cells to evaluating cell type composition of lysed whole tissues. This requires good and reproducible instrument setup and selection of high-performance controls to analyze and interpret the data. Controls are essential for any flow cytometry assay to reliably distinguish results from background variation and nonspecific effects.

Amerigo Scientific offers synthetic cells created by the intersection of biochemistry, polymer chemistry and high-precision manufacturing to address the reliance on and limitations around biologically-derived cellular controls. Our synthetic cells with high performance and consistency are used as ideal controls in flow cytometric analysis. With these synthetic cell controls, accurate, reliable and reproducible results as well as less time to determine results and more time to get results are achievable in you flow cytometry experiments.

FlowCytes® Synthetic White Blood Cells (WBC)

FlowCytes® synthetic WBC are shelf-stable synthetic cells that mimic the optical properties of human blood populations for unfixed or fixed lymphocytes, monocytes, and granulocytes. The products contain no biological material so they are safe to use in any environment and require no special disposal. There is no sample preparation needed, unlike traditional biological controls.

Features

• Easy to use and seamlessly fits into existing workflow
• No special handling and preparation, unlike biological controls
• Highly precise performance with minimal lot-to-lot variability
• Long shelf-life and stability at room temperature for convenient use
• Safe, non-biohazardous products that can be shipped anywhere

Applications

In flow cytometry, light is scattered by individual cells in a laser beam, and the light scatter properties of these cells distinguish cell populations. The measurement of forward scatter and side scatter allows for the discrimination of cells by size and the complexity or granularity of the cell, respectively. In addition, fluorescence can be used to measure extrinsic features such as specific protein expression and nucleic acid content using added reagents, such as fluorescent stains and antibodies.

FlowCytes® synthetic non-fixed or fixed WBC are intended as forward and side scatter controls for the identification and analysis of non-fixed or fixed whole blood populations and other cell types, respectively. The products can be used to calibrate and evaluate flow cytometry instrumentation or as a control in an assay to standardize gating. FlowCytes® are semi-transparent granules that resemble real cells instead of traditional, solid-core reagents. This makes FlowCytes® ideal for cell controls to calibrate, standardize and ensure the performance of instruments and assays.

Figure. Flow cytometric analysis of Purified PBMCs and FlowCytes® PBMC Mimics

TruCytes™ Synthetic Cells with Biomarkers

TruCytes™ are ready-to-use immunophenotyping controls for the positive detection of surface biomarkers, which are an extension of FlowCytes® products. These products are not derived from biospecimens so they are not biohazardous and require no special handling.

Our TruCytes™ synthetic cells are engineered with key surface antigen epitopes for standard immunophenotyping assays. A collection of biomarkers of TBNK controls (T cells, B cells, and NK cells) and stem cell controls can be precisely tuned and the antigen density also can be quantitatively modulated. Furthermore, TruCytes™ can be customized to precisely match rare diseases and any hematologic malignancy such as acute myelocytic leukemia (AML) and chronic lymphocytic leukemia (CLL).

Benefits

• Positive biomarker controls
• Accurate antigen density to ensure reliable results
• Superior consistency and reliability versus traditional controls (fresh primary cells, frozen controls, and lyophilized products)
• Exceptional product stability versus all biologically-derived cellular controls

Applications

In flow cytometry, current blood controls are commonly manufactured by using technology to combine human and animal cells or sourcing primary cells from active donors. Only less than 1% of blood diseases have available cellular controls. These biological controls are limited by high cost, batch-to-batch variability, the need for cell line maintenance, biohazardous transport and handling, and poor stability.

TruCytes™ are ready-to-use immunophenotyping controls that overcome the above limitations of biological controls and are used for the positive detection of specific surface biomarkers. They are ideal quality control (QC) and process control for assays that have measured readouts using flow cytometry.

Figure. TruCytes™ TBNK controls match the biochemical profile and optical properties of any target cell population, eliminating the drawbacks that traditionally come with using bio-based cellular controls.

Figure. TruCytes™ Indivadual Positive Population

SpectraComp® Synthetic Compensation Controls

SpectraComp® compensation beads are state-of-the-art synthetic cells that mimic the scatter profile of the lymphocyte populations, capture antibodies of multiple host species (mouse, rat, and hamster), and match the fluorescence spectra of stained cells.

Benefits

• Unparalleled high fluorescence response, which is greater than 10e5 mean fluorescence intensity (MFI)
• Conveniently works with multiple species of antibodies
• Exceptional performance in spectral analysis and unmixing
• Compatible with violet and UV fluorochromes
• Cell-like autofluorescence for superior signal-to-noise performance

Applications

The introduction of spectral flow cytometry has opened a wider range of applications and experimental possibilities, but it presents additional challenges related to compensation and analysis design. The reliance on more complex spectral flow experiments requires higher performance compensation reagents. In most flow cytometry experiments, polystyrene beads are used to perform compensation. Although the use of polystyrene beads is fast and convenient, there are a series of performance issues associated with polystyrene beads, including their inability to recapitulate the spectral profile of individual stained cells while also exhibiting high autofluorescence. This inevitably leads to distortion and under- or over-compensation, ultimately compromising the accuracy of the data.

SpectraComp® synthetic cells are designed as compensation controls to match the single staining performance of real cells. Staining SpectraComp® capture beads yields a positive and negative fluorescence curve, which will aid in resolving the performance of fluorophore and compensating for spectral overlap. SpectraComp® synthetic cells have the following advantages over polystyrene beads for applications:

• Spectral compensation controls with cell-like autofluorescence
• Accurate spectral unmixing for spectral analysis
• Easier experimental setup with less compensation analysis

Figure. Violet Dye Performance. SpectraComp® beads achieved cleaner separation of positive and negative populations when used in conjunction with violet dyes such as BV510.

ViaComp® Synthetic Viability Controls

ViaComp® viability controls are advanced 2-in-1 hydrogel beads and contain conveniently pre-mixed live and dead synthetic cell mimics. ViaComp® beads bind to both DNA intercalating dyes (7AAD, DAPI, etc.) and amine-reactive viability dyes to simulate the viability staining of cells. Our ViaComp® products are convenient, clean, and dependable viability controls that can be readily integrated into existing flow cytometry workflows.

Benefits

• Unique viability controls that bind to both DNA intercalating dyes and amine-reactive viability dyes
• Exceptionally positive peak signal (greater than 10e5 MFI) to make detection easy
• Minimal non-specific binding to the negative peak
• Ready-to-use pre-mixed negative and positive control beads

Applications

Viability staining is the process of distinguishing living and dead cell populations within a sample, which is important in experimental research and therapeutic applications of flow cytometry. In addition to emitting higher autofluorescence, dead cells also tend to nonspecifically bind to antibodies at high levels. Both factors can skew results towards an elevated population of false positives for one or multiple markers. This can be particularly detrimental for experiments evaluating a population that is weakly positive for the marker of interest. Therefore, the eliminating the effect of dead cells from analysis is fundamental to obtaining accurate data and ensuring consistency of results across multiple experiments. This necessitates a viability control that can clearly delineate where populations of live and dead cells would appear. Generally, viability controls are a mixture of live and dead cells stained with a differentiating dye. These dyes are usually classified as DNA-intercalating dyes and primary amine (protein) dyes. The two types of stains have different targets, but both rely on the increased cytoplasmic and nuclear membrane permeability of dead cells to more brightly label this population.

ViaComps® are designed as process and assay controls to match the viability staining of real cells. The combination of positive and negative binding beads yields positive and negative fluorescence peaks that will aid in identifying the live and dead cell populations. ViaComps® beads offer superior ease of use and reliable performance compared to traditional activity control reagents and methods.