Acoustic-assisted hydrodynamic focusing technology

Acoustic-assisted hydrodynamic focusing technology and advanced fluidics are designed to minimize clogging and effectively handle a broad range of cell types. This allows for a higher degree of data, detail, and throughput that enables processing of a large range of sample types, including large clumpy cells, samples with a low concentration of cells, and precious samples, more quickly and accurately than ever before with no loss in data quality.


Fluorescent proteins

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.

 

The Attune NxT Flow Cytometer was developed with fluorescent protein analysis in mind; it enables 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-sample-data-figure2

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 proteins (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.


CRISPR

A comparison of flow cytometry and other methods for analysis of CRISPR edited cells

The Attune NxT flow cytometer can be used quickly and effectively analyze the editing efficiency in CRISPR edited cells. Analysis by flow cytometry offers several advantages as compared to other methods of editing efficiency analysis:

 

  • 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 distinguish editing efficiencies of different cell types in mixed cell population.

Flow Cytometry Overlay Plots of Protein Knockdown in human PBMCs at the TCR alpha/beta, B2M and PD-1 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, 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. The antibodies used were TCR alpha/beta Monoclonal Antibody (IP26), PE, eBioscience, beta-2 Microglobulin Monoclonal Antibody (B2M-01), FITC, and CD279 (PD-1) Monoclonal Antibody (eBioJ105 (J105)), APC-eFluor 780, eBioscience

Analysis of CRISPR edited cells using the Attune™ NxT 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 samples processing time are a few of the benefits of using flow cytometry for analysis of gene editing for flow cytometry.

Analysis of Multiplexed CRISPR Edits by Flow Cytometry

Flow cytometry a high-throughput, rapid, and accurate method for quantify 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.

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 function protein knockdown at each locus. Samples were run on the Attune NxT Flow Cytometer and CytKick Autosampler. The overlay plots above were generated the Attune NxT software. Each histogram is overlayed with the non-neon control. Antibodies used were TCR alpha/beta Monoclonal Antibody (IP26), PE, eBioscience, beta-2 Microglobulin Monoclonal Antibody (B2M-01), FITC, and CD47 Monoclonal Antibody (B6H12), APC, eBioscience

 

Gating Strategy and Analysis of Edited Cells Using the Attune NxT Software

 

The multiplexed well was analyzed by gating on live, single cells and protein knockdown gates drawn using the negative control (non-neon sample). To determine proportion of cells with multiplexed CRISPR-mediated protein knockdown, a derived gate (Triple KO) was created using Boolean logic formula (NOT "B2M+") AND (NOT "CD47+") AND (NOT "TRAC+") AND "Singlets" AND "Cells".

Proportion of Cells edited at TCR alpha/beta, PD-1 and B2M Loci

 

The total cell population was plotted and back gated with the “Triple KO” gate shown in red. The proportion of cells with protein knockdown for all 3 loci was determined using the Attune NxT plot statistics function.


Immuno-oncology

Achieve a reliable measure of accuracy for rare events

With the Attune NxT Flow Cytometer 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.


Immunophenotyping

13-parameter immunophenotyping of human lysed whole blood 

Flow cytometry is the method of choice for identifying cells within complex populations, as it allows for multi parameter analysis of thousands to millions of cells in a short time. Strong signal separation in the Attune NxT Flow Cytometer shows excellent separation 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 multi-color 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.

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

 

Dead cells were excluded from the analysis by gating on live cells in a dot plot (A). CD45-positive cells were gated on to select the leukocyte population from the lysed whole blood (B). Lymphocytes and monocytes were gated based on forward and side scatter profiles (C). Monocytes are found above lymphocytes based on scatter profiles and express both CD14 and CD33 (D). Within the lymphocyte gate T cells can be isolated based on their expression of CD3 (F) and further subdivided into CD4 (T helper cells) and CD8 (cytotoxic T cells) subpopulations (G). B cells can be further characterized by HLA-DR and CD45RA expression (E). In addition, regulatory T cells express CD4 and CD25 (J). CD62L identifies naive (TN) CD4 and CD8 T cells, whereas HLA-DR is expressed by activated T cells (TA) (H,K). NK cells can be identified as they lack B cell (CD19) and T cell (CD3) markers and express CD56 (I).

 

Download the application note for more information.


Microbiology

Quick and accurate detection of wastewater

On traditional flow cytometers, very dilute samples can take a long time to acquire due to slower flow rates. The Attune NxT Flow Cytometer can run very dilute samples quickly (below).

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.


Plant ploidy

Rapid and 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 in plant homogenates, and the use of flow cytometry in plant biology has increased rapidly. The Attune NxT Flow Cytometer is well suited for DNA content evaluation. Any of the standard configurations may be used, including the most affordable single-laser system.

5-rapid-and-accurate-analysis-of-nuclear-DNA2

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.

 

Download the application note for more information.


Platelets

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. 

 

The Attune NxT Flow Cytometer, along with the Attune NxT No-Wash, No-Lyse Filter Kit for violet laser SSC detection, offers 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. 

5-detection-of-platelets-in-whole-blood2

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

 

(A, B) RBCs, 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 detailed methods and results using two different methods for the detection of platelets in human blood using the system.


Stem 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. The Attune NxT Flow Cytometer is 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.

 

5-stem-cells-and-cardiomyocytesv2

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.


T cells

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 7 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.  

5-T-cell-backbone-panelv2

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 (B) or with PPD of Mycobacterium tuberculosis (C) or 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, (E) followed by gating on single cells, (F, G) and then dump channel– CD3+ CD4+ cells. 

 

Download the application note for more information. 

Detection of murine regulatory T cells

Interest in regulatory T cells 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 NxT Flow Cytometer shows excellent separation 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. 

 

(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.

 

Download the application note for more information.