Attune NxT Flow Cytometer Sample Data

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.

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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

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.

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 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).

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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.

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. 

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