Attune Flow Cytometers Sample Data

• Doublet discrimination with image data
• Clumpy CEN cells
• Cell culture QC

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 (Manual Singlets) 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).

Aged whole human blood (AllCells) lysed with ammonium chloride lysis buffer. Image processing was done using the Cells_Half_Resolution_v22 model. Statistics shown are % gated.

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.

Accurate gating for sticky CEN cells. CEN cells were stained with PI per manufacturer directions (BD) and acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. On a PI histogram, gates were originally drawn to include the shoulders on both sides of each peak (CEN gating strategy 1, top center), expecting that gate I would contain singlets, gate II doublets, and so on. However, images of doublets (left) were captured within gate I. Moving gate boundaries to the left to exclude the right shoulders for each peak (CEN gating strategy 2, right) effectively classified both single cells and aggregates within the correct gate.

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.

Contamination of a Ramos cell culture. Ramos (lymphoma) cells in culture showed reduced cell counts and survival during a routine passage quality check, despite appearing confluent. Further evaluation showed microbial contamination, confirmed by imaging and backgating on the Attune CytPix Flow Cytometer (A,B). Early signs of this contamination (C) had initially been dismissed as debris.

• Immuno-oncology rare event analysis
• CAR T immunotherapy

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).

Detection of rare ILC2 population in PBMCs
(A) Labeling of 1 x 10⁶ PBMCs resuspended in 100 μL PBS (+10% FBS). The antibodies used were a lineage cocktail containing CD2, CD3, CD14, CD16, CD19, CD56, and CD235a conjugated to Invitrogen FITC, CD123-FITC, and CRTH2-Alexa Fluor 647 conjugates. The ILC2 cells are then defined as the lineage (BL1)-negative, CRTh2 (RL1)-positive populations. (B) CRTH2 cells expressing the chemoattractant receptor–homologous molecule expressed on Th2 cells. CRTH2 is a seven-transmembrane protein coupled with heterotrimeric G proteins. CRTH2 is the prostaglandin D2 receptor and is expressed by Th2 cells, eosinophils, and basophils. CD294 prevents the apoptosis of Th2 cells and mediates the chemotaxis of CRTH2-expressing cells to the sites of allergic inflammation, such as the asthmatic lung. (C) The ILC2 cells are defined as lineage-negative and CRTH2-positive. In this example, the ILC2 population is 0.016% of the parent gate. Data courtesy David Cousins, University of Leicester.

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.

Visualization of CAR T cells targeting lymphoma cells. CAR T cell and Ramos cells were labeled with CellTrace Far Red and CellTrace Violet respectively and incubated at a 1:1 ratio for 1 hour at 37°C. Unfiltered samples were acquired on the Attune CytPix Flow Cytometer at 200 µL/minute, >8 x 10⁵ cells/mL. Images of quadrants Q1 (top left), Q4 (bottom right), and Q3 (bottom left) show individual Ramos cells, CAR T cells, and debris, respectively. Images from quadrant Q2 (positive for both stains, top right) reveal both cell types fused together, acquired as a single event as the CAR T cells engulf the Ramos cells.

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. 

Visualization of CAR-T cells targeting lymphoma cells. CAR-T and Ramos cells were labeled with CellTrace Far Red and Violet respectively and incubated at a 1:1 ratio for 1 hour at 37°C. Unfiltered samples were acquired on the Attune CytPix flow cytometer at 200 µL/minute, >8 x 10⁵ cells/mL. Images of quadrants Q1 (top left), Q3 (bottom right), and Q4 (bottom left) show individual Ramos cells, CAR T cells, and debris, respectively. Images from quadrant Q2 (positive for both stains, top right) reveal both cell types fused together, acquired as a single event as the CAR-T cells engulf the Ramos cells. Percentages are % gated.

In the cell image galleries, annotated events are outlined in black with yellow dots indicating center positioning. Image processing was done using the Cells Half Resolution model by analyzing circularity versus skewness of intensity. This allows us to differentiate between attached cell interactions (between CAR-T and Ramos cells) and detached where cells are in the same field of view but not showing cell-to-cell interactions.

Colorectal cancer cells were spiked into healthy human PBMCs at a rate of 1,000 cells per sample. All samples prepared by Amsterdam UMC. Statistics shown are cell counts. Image processing was done using the Cells_Half_Resolution_v22 model.

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.

 SARS-CoV-2 Nucleoprotein staining of infected 293T-ACE2 cells. (A) Uninfected cells and (B) SARS-CoV-2 infected 293T-ACE2 cells after 36 hours at an MOI of 0.1. Cells were prepared using standard protocols and stained using a SARS coronavirus nucleoprotein monoclonal antibody (clone 1C7C7) and anti-mouse secondary antibody conjugated to PE (Cat. No. P852). Data was collected using an Attune NxT flow cytometer equipped with autosampler. Analysis was performed in FCS Express 7.

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.

Flow cytometry analysis of transcription factors during cardiomyocyte differentiation. Two-parameter plots representing staining profiles for Oct4 and Nkx2.5 in H9 hPSC cells during cardiomyocyte differentiation. All plots were gated on singlet cells. (A) At day 1, nearly all cells are Oct4⁺ and Nkx2.5^–, consistent with a pluripotent state. (B–J) During the time course of differentiation, with data shown for each day of differentiation, cells lose Oct4 expression and begin to express the cardiac marker Nkx2.5. The precedence-density plot display is used, with the red-colored population representing Nkx2.5⁺ cells, and the green-colored population representing Oct4⁺ cells.

• Microbiology
• Imaging-enhanced microbiology

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.

Discrimination of two E. coli CFU types. E. coli cells were incubated overnight at 37ºC followed by 3 days at 4ºC. Samples were acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the images, two types of CFUs were identified: (A) short colonies resembling single cells and (B) elongated structures with incomplete fission rings. Representative images from each population are shown. Backgating on the selected images demonstrated that the two populations are distinct on FSC vs SSC dot plots (orange dots, left).

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.

Data were collected from plant nuclei prepared from A. thaliana Col-1 leaf tissue and labeled with FxCycle PI/RNase Staining Solution using the 532 nm laser. (A) Biparametric density plot of side scatter vs. FxCycle PI/RNase fluorescence, with a scatter gate surrounding the fluorescent nuclei. (B) Biparametric density plot of FxCycle PI/RNase fluorescence to gate on singlet nuclei. (C) Logarithmic histogram of FxCycle PI/RNase fluorescence of nuclei-gated population, showing multiple peaks corresponding to 2C, 4C, 8C, and 16C nuclei.
 

Download the application note for more information.

For microbiology data showing two distinct types of E. coli colony-forming units (CFUs), see "Imaging-enhanced microbiology" in the Imaging-enhanced flow cytometry section above.

• Analysis of multiplexed CRISPR edits by flow cytometry
• Analysis of CRISPR edited cells

Screening for protein knockdown efficiency at multiple loci. Human peripheral blood mononuclear cell (PBMCs) were cultured, and T cells subsequently activated using Dynabeads Human T-Activator CD3/CD28 kit. Cells were then edited using Invitrogen TrueGuide Synthetic gRNA, TrueCut Cas9 Protein v2 and the Invitrogen Neon Transfection System. gRNAs targeting the human T cell receptor, Beta-2-Microglobulin and CD47 genes were designed using the Invitrogen TrueDesign Genome Editor tool. Cells were analyzed for editing efficiency 72 hours post transfection by flow cytometry analysis, as measured by functional protein knockdown at each locus. Samples were run on the Attune NxT Flow Cytometer and CytKick Autosampler. The overlay plots above were generated by Attune Cytometric Software. Each histogram is overlayed with the non-neon control. Monoclonal antibodies used were TCR alpha/beta (IP26) PE (eBioscience), Beta-2 Microglobulin (B2M-01) FITC, and CD279 (PD-1) (eBioJ105 (J105)) APC-eFluor 780, (eBioscience).

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:

• Provides efficiency data of functional protein knockdown.
• Provides single cell, multiparameter knockdown efficiency analysis, i.e. the proportion of cells that are simultaneously edited at multiple loci.
• Adds additional parameters for analysis, such as proliferation, viability, and differential editing efficiencies of different cell types in mixed cell populations.

Flow cytometry overlay plots of protein knockdown in human PBMCs at the TCR alpha/beta, B2M and PD-1 loci. Human peripheral blood mononuclear cells (PBMCs) were cultured, and T cells subsequently activated using Dynabeads Human T-Activator CD3/CD28 kit. Cells were then edited using Invitrogen TrueGuide Synthetic gRNA, TrueCut Cas9 Protein v2 and the Invitrogen Neon Transfection System. gRNAs targeting the human T cell receptor, Beta-2-Microglobulin and CD47 genes were designed using the Invitrogen TrueDesign Genome Editor tool. Cells were analyzed for editing efficiency 72 hours post transfection by flow cytometry, next-gen sequencing, Sanger sequencing analysis and the genomic detection cleavage assay. The Attune NxT software was used for all figures and data analysis. Each histogram is overlayed with the non-neon treated control and each figure is data collected from a single well. Monoclonal antibodies used were TCR alpha/beta (IP26) PE (eBioscience), Beta-2 Microglobulin (B2M-01) FITC, and CD279 (PD-1) (eBioJ105 (J105)) APC-eFluor 780, (eBioscience).

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.

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.

Detection of multiple fluorescent proteins expressed in the same cell. 293FT cells were transfected with two plasmids, either by sequential delivery of each plasmid separately (top panels), or in 1:1 (w/w) mixes (bottom panels), using Invitrogen Lipofectamine 3000 reagent. Transfected cells were grown for 48 hr prior to harvest and analysis by flow cytometry. Samples were acquired using the Attune NxT Flow Cytometer at a flow rate of 100 μL/min, and a minimum of 15,000 events were collected for each sample. All major cell populations are detected: cells expressing one of the fluorescent proteins, both fluorescent proteins and neither fluorescent protein (percentages are indicated on the plots). Cells expressing the Fluorescent Proteins are easily distinguished from non–fluorescent proteins-expressing cells. (A) The 405 nm and 561 nm lasers were used for excitation of TagBFP and mOrange2, respectively. (B) The 405 nm and 561 nm lasers were used for excitation of TagBFP and mKate, respectively. (C) The 488 nm and 561 nm lasers were used for excitation of emGFP and mKate, respectively.
 

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