Sample data across a wide range of applications shows how acoustic-assisted hydrodynamic focusing technology allows researchers to process more sample types—including large clumpy cells and samples with low cell concentrations—more quickly and accurately than before. The high-speed camera in the Attune CytPix Flow Cytometer enables a variety of new applications that combine cell morphology data from imaging with size, granularity, and multiplexed protein expression data from cytometry.
Potential applications of an imaging-enhanced flow cytometer include almost any study that can benefit from understanding the morphology of each cell population identified.
Morphological information from images can add to the richness of apoptosis analysis. This apoptosis experiment using Annexin V and propidium iodide (PI) added cell imaging to characterize cells in each population to reveal morphologically distinct features. These insights could not have been gained from flow cytometry data alone.
Imaging can be used to confirm and adjust gates to include only single cells of interest. Chicken erythrocyte nuclei (CEN) cells are notoriously sticky and tend to clump into doublets or other aggregates. Researchers often identify these aggregates using propidium iodide (PI) assays in which successive peaks correspond to the number of cells in an event. But imaging revealed that next-level aggregates begin to appear in the right shoulders of the preceding peaks. For example, the right shoulder of peak I (assumed to include only singlets) contained many doublets. Tightening the gates successfully removed the unwanted doublets and shifted them appropriately into the next gate.
Even robust manual singlet gating is error-prone and remains a subjective decision point in almost all flow cytometry assays. Imaging can be used to confirm and adjust gates to include only single cells of interest.
Here, an experienced user has gated singlets confidently. After evaluating their singlet gate (ManualSinglets) using derived parameters from CytPix instrument images, we can see that this gate contains >4% aggregates.
Perhaps most importantly, these events contain cells of clearly different phenotypes which may have led to incorrect conclusions regarding double positive events (especially in rare populations).
Imaging can show evidence of cell function, including interactions among immune cells. Engineered CAR T immunotherapy cells were co-incubated with Ramos (lymphoma) cells, stained, acquired, and imaged on the Attune CytPix Flow Cytometer. Images from quadrant Q2 (positive for both stains, acquired as a single event) show the CAR T cells visibly targeting the Ramos cells, clear evidence of engineered immune cell potency.
We previously demonstrated the power of imaging CAR-T/Ramos cell interactions. Let’s look at just the population of greatest interest, the double positive events, to learn more. We can now use extended image-derived parameters (circularity vs skewness of intensity) to further refine this population, increasing data robustness. Here we show that by using the image-derived parameters, we can distinguish cell-cell interactions from coincident events more accurately.
E. coli cells incubated over time develop into two types of colony-forming units (CFUs): short CFUs that resemble single cells, and elongated structures with incomplete fission rings, representing incomplete constriction at each approximate cell length. Neither a traditional singlet gate (SSC-A vs SSC-H) nor a fluorescence gate (SSC vs nucleated stain) sufficiently separates these populations. But with the Attune CytPix imaging-enhanced flow cytometer, you can view and group the images and then gate the CFU types based on their morphological characteristics.
Adding rapid imaging to quality control (QC) workflows can detect and identify cell culture issues early in the process. In one lab, a routine passage check of a Ramos (lymphoma) cell culture observed reduced cell counts and survival despite appearing confluent. Further investigation revealed substantial microbial contamination, but when and where did it begin?
Because the cell line had previously been analyzed on the Attune CytPix Flow Cytometer, the researchers went back to the images and were able to document the microbial infection at least five days earlier. At that time, the early signs were dismissed as debris, but the retrospective evaluation demonstrated shared characteristics with the problematic cells in culture. Tracing the infection helped the lab establish additional laboratory procedures for screening and protection of assay-critical cell lines.
Cell imaging can be used to improve immunophenotyping experiments and train new users. Cell outline and measurement tools integrated into the imaging software supplement imaging and flow cytometry to characterize cell populations and set and confirm gates.
To demonstrate the capacity of the image analysis software to enhance separation of rare cells from mixed cell populations, we spiked a peripheral blood sample with 1,000 colorectal cancer cells. To detect these very rare events, we collected over 4.5 million events (500 µL/minute run rate). Only events double positive for markers which identified the target cells (EpCAM & EGFR) were imaged. By using the Attune CytPix to image these double positive events, we found that many of them were not single cancer cells but were instead debris/aggregates of unexpected morphology.
By combining derived parameters (diameter, circularity, and intensity parameters were utilized) with existing fluorescence data (such as the APC-labeled EpCAM staining), the user was able to improve their gating of target cells. This gating strategy yielded more accurate quantitation of the EpCAM+EGFR+ cells than fluorescence alone.
Showing appropriate detachment of magnetic selection beads is often a critical but time-consuming step in cell and gene therapy workflows. Extended image parameters makes accurate identification of beads more efficient than ever. Here, we see singlet and aggregate beads separated from single and aggregate cell-containing events.
To analyze further, the Attune CytPix Flow Cytometer was set to image CD45+ events, presumed to represent WBCs. The images demonstrated that some of these events actually represent clusters, small platelets, dark RBCs, or combinations of cells analyzed as single events. What appeared to be a homogeneous population is actually more diverse—an insight that should be considered when interpreting results.
Because leukocytes (white blood cells or WBCs) comprise only about 0.1% of whole blood cells, the more populous erythrocytes (red blood cells or RBCs) are often lysed to separate them out. However, this may also lyse or alter some WBCs. In an application note, we validated a no wash/no lyse method of distinguishing unlysed RBCs, WBCs, and platelets, using the property that hemoglobin in RBCs readily absorbs violet (405 nm) light, while WBCs and platelets do not. This shifts the RBCs to the right on a blue vs violet side scatter (SSC) plot, using the Attune CytPix No-Wash, No-Lyse Filter Kit to allow dual-laser light scatter detection (Panel A).
Staining WBCs for CD45 expression, however, shows that some WBCs (pale blue in the dot plot) appear in the erythrocyte gate. To analyze further, the Attune CytPix Flow Cytometer was set to image CD45+ events, presumed to represent WBCs. The images (Panel B) demonstrated that some of these events (with dots backgated in purple) actually represent clusters, small platelets, dark RBCs, or combinations of cells analyzed as single events. What appeared to be a homogeneous population is actually more diverse—an insight that should be considered when interpreting results.
For a no wash/no lyse data example, see Immunophenotyping later on this page.
Attune flow cytometers offer a fast, easy, and accurate platform to measure protein and gene expression. This includes viral proteins expressed in infected host cells.
In the data below, researchers were able to measure SARS-CoV-2 nucleoprotein expression in cultured human cells before and after exposure to SARS-CoV-2, the novel coronavirus that causes COVID-19 disease.
"[We] have built robust immunophenotyping panels to identify infected cell types in primary human tissue systems. The high throughput nature of the plate reader allows us to conduct more complicated and informative experiments and has fundamentally changed the way that we conduct our research."
Josh Acklin, Researcher
Icahn School of Medicine at Mount Sinai
Today fluorescent proteins are widely used in the investigation of gene expression as well as protein localization, translocation, and trafficking within live cells. More advanced techniques include assessment of protein–protein interactions and spatial relationships of proteins in live cells using fluorescence resonance energy transfer (FRET) techniques and fluorescence lifetime imaging microscopy (FLIM).
The simultaneous detection of multiple fluorescent proteins in the same cell has traditionally been more difficult than the detection of multiple fluorophore-labeled antibodies. This is in part because fluorescent proteins have a different, broader emission spectrum than the traditional cell dyes and fluorophores used in the labeling of antibodies.
Attune Flow Cytometers were developed with fluorescent protein analysis in mind; they enable easy and accurate analysis of multiple fluorescent proteins and fluorescently labeled antibodies (separately or in combination), with configurations allowing up to 4 lasers and 16 detection channels.
Attune flow cytometers can be used to quickly and effectively analyze the editing efficiency in CRISPR edited cells. Analysis by flow cytometry offers several advantages as compared to other methods of analyzing editing efficiency:
Analysis of CRISPR edited cells using Attune Flow Cytometers provides accurate and rapid quantification of editing efficiency, and is particularly beneficial when multiplexing multiple CRISPR gRNAs. Single cell analysis, functional knockout efficacy, quick actionable data, and minimal sample processing time are a few of the benefits of using flow cytometry for analysis of gene editing.
Flow cytometry is a high-throughput, rapid, and accurate method for quantifying functional protein knockdown in CRISPR-edited cell populations. Flow cytometry is particularly beneficial when analyzing cell populations edited with multiple gRNAs, as it provides the ability to ascertain single cell protein knockdown efficiency at multiple loci, without the need for clonal isolation.
The workflow is streamlined, requires minimal reagents and hands-on time, and provides rapid and accurate results.
With Attune Flow Cytometers you can achieve a reliable measure of accuracy for detection of cell populations comprising less than 1% of the total cells by easily running large sample volumes in a fraction of the time without the need to concentrate your sample (below).
Flow cytometry is the method of choice for identifying cells within complex populations, as it allows for multiparameter analysis of thousands to millions of cells in a short time. Strong signal separation in the Attune Flow Cytometer shows excellent resolution of cell populations into subsets for immunophenotyping. A wide range of reagent choices, as well as the system’s automated compensation module, 4 spatially separated lasers, and 14 color choices help simplify multicolor panel design.
The data below describes 13-color immunophenotyping analysis of stained human whole blood using a stain/lyse protocol on the Attune NxT Flow Cytometer. Lymphocyte, monocyte and granulocyte populations were distinguished with forward scatter (FSC) and side scatter (SSC); and monocyte, T cell, B cell and NK populations were identified using fluorescent antibodies against surface antigens specific for the different immunological populations.
On traditional flow cytometers, very dilute samples can take a long time to acquire due to slower flow rates. Attune Flow Cytometers can run very dilute samples quickly (below).
Providing a simple, fast, accurate, and reliable methodology, flow cytometry has become the method of choice to determine C-values (amount of nuclear DNA content) in plant homogenates, and the use of flow cytometry in plant biology has increased rapidly. Attune Flow Cytometers are well suited for DNA content evaluation. Any of the standard configurations may be used, including the most affordable single-laser system.
Resting platelets are the smallest cellular component of peripheral blood. Upon activation, platelets undergo rapid changes in cell surface receptor expression that lead to altered adhesive properties and changes in morphology that promote the formation of a platelet plug at the site of vascular disruption. These properties can make the interrogation of platelets by flow cytometry challenging, especially in the context of light scatter detection.
Attune Flow Cytometers, along with the Attune NxT No-Wash, No-Lyse Filter Kit or Attune CytPix No-Wash, No-Lyse Filter Kit for violet laser SSC detection, offer a robust assay for detecting platelets in whole blood without sample manipulation. The system’s acoustic focusing technology empowers research with unmatched speed (up to 10 times faster than the traditional cytometers), thereby greatly reducing the assay time.
The ability to direct human pluripotent stem cells (hPSCs) toward differentiated cell phenotypes offers tremendous potential for personalized and regenerative medicine. Attune Flow Cytometers are ideally suited for use with fragile and large cell types like stem cells and cardiomyocytes (below). Engineered to actively resist clogging, a syringe-driven system and larger flow cell help prevent the loss of precious sample and is drastically less susceptible to clogs.
Memory antigen-specific CD4 T cells are quite rare in the circulating blood, with frequency ranging from 1 in 100 to less than 1 in 100,000 depending on the antigen and normal range variation. Flow cytometry is an effective technology to monitor and identify rare cells among a mixed population of different cells types. Not only is it capable of rapidly identifying unique cell types, but it can also be used to analyze many other phenotypic features at the single-cell level, making it a valuable tool for understanding the immune system.
In this study, a viability dye (Invitrogen LIVE/DEAD Fixable Near-IR Dead Cell Stain) and seven antibodies, including CD137 and CD69, were used as a backbone panel to identify antigen-specific CD4 T cells using the Attune NxT Flow Cytometer, 4-laser configuration.
Interest in regulatory T cells (Tregs) has been accelerated by evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in research associated with autoimmunity, infectious agents, and cancer.
Strong signal separation in the Attune Flow Cytometer shows excellent resolution of cell populations into subsets for immunophenotyping. This 3-color immunophenotyping analysis of stained mouse splenocytes using the Foxp3 Transcription Factor Staining Buffer Kit shows excellent cell population resolution for mouse regulatory T cells consisting of both surface and intracellular markers.
For Research Use Only. Not for use in diagnostic procedures.