Attune Flow Cytometers Sample Data

Acoustic focusing and a high-speed camera result in rich data detail

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 multiplexed protein expression data from cytometry.

Basics of flow cytometry analysis: sample quality and cell health

Doublet discrimination with image data

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.

representative flow plots and cell images showing user error in gating single cells

Clumpy CEN cells

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.

Brightfield images can reveal need to reset gating

Cell culture QC

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.

Brightfield image of Ramos cell contamination

Apoptosis analysis enhanced with imaging

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.

Morphological characteristics of apoptotic cells

Jurkat cells were incubated with 10 µM camptothecin for 4 hours at 37ºC to induce apoptosis. Samples were stained with Annexin V and PI and acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the singlet population, gating strategies identified three cell subpopulations. About 50% of apoptotic live cells (Annexin V+PI–, bottom right) showed some form of apoptotic body such as blebs. About 25% of apoptotic dead cells (Annexin V+PI+, top right) showed increased cell surface granularity, and there were more partial cells. About 10% of healthy cells (Annexin V–, bottom left) showed apoptotic bodies (though not as severe as those observed among Annexin V+ cells). These healthy cells were also morphologically diverse and included some doublets despite upstream singlet gating. Morphological features in the images are indicated by black arrows.

 

PI- and annexin-stained Jurkat cells to detect apoptosis

Immunology and immuno-oncology

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.

 

13-parameter immunophenotyping of human lysed whole blood

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

Gating strategy for 13-color immunophenotyping analysis of stained human whole blood using a stain/lyse protocol

Human whole blood cells were stained as described in the application note and acquired and analyzed on the Attune NxT Flow Cytometer. (A) Dead cells were excluded from the analysis by gating on live (propidium iodide-) cells in a dot plot. (B) CD45+ gating was used to select the leukocyte population from the lysed whole blood. (C) Lymphocytes and monocytes were identified based on forward and side scatter profiles. (D) Monocytes are found above lymphocytes on the scatter plot and express both CD14 and CD33. (F) Within the lymphocyte gate, immune cells can be subdivided based on their expression of CD3 (T cells), CD19 (B cells), or neither (NK cells). (E) B cells can be further characterized by HLA-DR and CD45RA expression. (G) T cells can be further subdivided into CD4+ (T helper cells) and CD8+ (cytotoxic T cells) subpopulations, while (J) regulatory T cells express CD4 and CD25. (H, K) CD62L identifies naive (TN) CD4 and CD8 T cells, while HLA-DR is expressed by activated T cells (TA). (I) Finally, NK cells lack B cell and T cell markers (CD19–CD3–) and express CD56.

Download the application note for more information.

Gating strategy for 13-color immunophenotyping analysis of stained human whole blood using a stain/lyse protocol

Imaging-enhanced immunophenotyping

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.

Imaging and measurement of PBMC cell populations

Cryopreserved peripheral blood mononuclear cells (PBMCs) were restored, rested for 1 hour at 37°C, and stained for CD3, CD19, and CD14 to distinguish T cells, B cells, and monocytes respectively. Samples were acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the singlet population, gating strategies identified T cells (blue), B cells (rose), and monocytes (gray), shown backgated on an FSC vs SSC plot, and images of each subset were displayed. Integrated cell outline and measurement tools (red) calculated the areas of a representative monocyte (101.26 µm2), T cell (38.08 µm2), and B cell (64.77 µm2).

Brightfield images measured to confirm lymphocyte populations

No wash/no lyse leukocyte analysis with imaging

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.

No wash/no lyse leukocyte analysis with imaging

Cells were acquired from 24-hour-old blood by dilution in 1 mM EDTA (<1:4000). Samples were stained with FITC anti-CD45 using a no wash/no lyse protocol and acquired on an Attune CytPix Flow Cytometer equipped with the Attune CytPix No-Wash, No-Lyse Filter Kit to allow dual-laser light scatter detection. (A) A dot plot of blue vs violet SSC shows separate regions for erythrocytes vs platelets and leukocytes. However, some CD45+ events (pale blue) appear in the erythrocyte region. (B) Gating and imaging on only CD45+ events shows that some events (purple dots) represent clusters, platelets, RBCs, or combinations of these or other cells.

Attune CytPix brightfield images can be used with no wash no lyse protocol to accurately gate WBCs

Magnetic bead stimulated human PBMCs: imaging-enhanced analysis

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.

Human PBMCs stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 Beads. Image processing was done using the Cells_Full_Resolution_v21 model.

Annotated events are outlined in black with yellow dots indicating center positioning. Statistics shown are % gated.

representative data and image galleries of bead and cell aggregate events separated from single cells and single beads

T cell backbone panel

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.  

Five-color backbone panel for antigen-specific circulating CD4 T cells.

(A) Two-parameter plots showing expression of CD69 and CD137 are shown for undiluted whole blood from a healthy donor that was cultured for 24 hours without antigen or (B) with PPD of Mycobacterium tuberculosis or (C) a CMV cell lysate antigen preparation. The cells were then harvested and stained with backbone panel antibodies including CD137, CD69, CD3, CD4, CD19, CD16, and CD14 with LIVE/DEAD Fixable Near-IR Dead Cell Stain for viability and analyzed on the Attune NxT Flow Cytometer. (D) Lymphocytes were identified using light scatter gates, followed by gating on (E) single cells, and then (F, G) dump channel– CD3+ CD4+ cells.

 

Download the application note for more information. 

Identifying antigen specific CD4 T cells

Detection of murine regulatory T cells

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. 

Detection of murine regulatory T cells on the Attune NxT Flow Cytometer.

Detection of murine regulatory T cells on the Attune NxT Flow Cytometer

 

(A) Bivariate dot plot depicting the CD4+ Foxp3+ regulatory T cell population (gated) present in mouse spleen (A, left panel) compared to isotype control (A, right panel). Cells were gated on lymphocytes based on FSC/SSC profile. (B) CD4+ T cells were gated and analyzed for CD25 and Foxp3 expression. The majority of murine regulatory T cells co-express the transcription factor Foxp3 and the cell surface marker CD25.
 

View the application note for more information. 

Detection of platelets in whole blood

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. 

Dual laser blue (488 nm) and violet (405 nm) laser SSC with intact whole blood (no-wash/no-lyse).

(A, B) Red blood cells (RBCs), white blood cells (WBCs), and platelets are separated on the basis of light scatter only by using a combination of blue and violet laser SSC analysis. Hemoglobin in RBCs readily absorbs light at 405 nm, shifting the RBC population to the right by reducing the SSC for RBCs in the violet SSC channel relative to leukocytes and platelets. Dual FSC and SSC threshold is set low enough to show instrument noise, ensuring the full platelet population is visualized. (C) Using the gate that includes WBCs and platelets, a standard plot of FSC vs. 488 nm SSC can be used to distinguish the platelet population from the WBCs with regions created around the two populations. (D) Using color-backgating on the same plot as previously shown in (A), the RBC population is colored red, the platelet population is colored green, and the WBC population is colored blue, while the noise is black. The three main WBC populations of lymphocytes, monocytes, and granulocytes can be distinguished. (E) Placing regions around the RBC, WBC, and platelet populations show the dominant cell type in whole blood is the RBC, while the WBC and the platelets are relatively rare events.


Download the application note for more information.

gating strategy detecting platelets in whole blood

Inmuno-oncology rare event analysis

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.

Gating strategy to identify rare cell populations

CAR-T immunotherapy and cell-to-cell interactions

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.

Brightfield images allow confirmation of CAR T-cell actions

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.

representative data of cell-to-cell interactions and coincident events in a processed flow sample

Other applications

Imaging-enhanced rare cell separation in complex mixed samples

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. 

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.

representative data of unexpected cell aggregates within a double positive gate

Detecting SARS-CoV-2 infected cells

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.

Flow cytometry dot plots showing side scatter vs fluorescence in uninfected and SARS-CoV-2 infected cells

Stem cells and cardiomyocytes

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.

Identifying pluripotent stem cells

Microbiology

Quick and accurate detection of wastewater pathogens

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).

Live and dead bacteria in a wastewater sample

Analysis of bacteria in treated municipal wastewater on the Attune NxT Flow Cytometer

 

A 3 mL sample of municipal wastewater was labeled with the Invitrogen LIVE/DEAD BacLight Bacterial Viability Kit and analyzed on the Attune NxT Flow Cytometer at a flow rate of 1 mL/min, which allowed quick analysis of the sample and accurate detection of very small quantities of bacteria. Concentrations of the live and dead bacteria where determined without using reference counting beads. The two-parameter dot plot (propidium iodide vs. SYTO 9 fluorescence), with the live (green) and dead (red) bacterial populations are well separated; the statistics table displays the concentration measurements for the labeled bacteria. Wastewater may also include small eukaryotes and types of bacteria that are potentially viable but nonculturable, each of which may also be labeled with the dyes; the grey dots represent debris found in the wastewater.

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).

Brightfield images confirm short E.coli CFUs

 

Rapid and fast accurate analysis of nuclear DNA

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.

flow cytometry dot plots showing applied gates and final histogram of 2C, 4C, 8C, and 16C populations

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

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.

 

Live and dead bacteria in a wastewater sample

Workflow for Analysis of CRISPR edited cells by flow cytometry

 

Quantifying CRISPR mediated protein knockdown by flow cytometry requires only a validated antibody for the protein target of interest and a flow cytometer.

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).

Screening for protein knockdown efficiency at multiple loci

Analysis of CRISPR edited cell

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).

Flow cytometry overlay plots of protein knockdown in human PBMCs at the TCR alpha/beta, B2M and PD-1 loci

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.

Detection of fluorescent proteins

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.

Download the application note for more information.

Detection of multiple fluorescent proteins expressed in the same cell

For Research Use Only. Not for use in diagnostic procedures.