Immunophenotyping With the Attune® Cytometer

The Attune® Acoustic Focusing Flow Cytometer shows excellent separation of cell populations into subsets for immunophenotyping experiments (up to six colors).

  • Six-color detection with minimal compensation
  • Strong signal separation for more data clarity
  • Less need for difficult tandem dyes


Below figures demonstrate six-color immunophenotyping analysis on the Attune® Acoustic Focusing Cytometer with both blue/violet and blue/red laser configurations.

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Immunophenotyping With Blue/Violet & Blue/Red Configurations

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Figure 1. Six-color immunophenotyping analysis on the Attune® Acoustic Focusing Cytometer with blue/violet laseroption.
Normal human blood cells were labeled for six-color Immunophenotyping with the following directly labeled mouse anti-human antibody conjugates: CD45-Pacific Orange™, CD3-FITC,CD8-Pacific Blue™, CD56-R-PE, CD19-TRI-COLOR® (all from Life Technologies), and CD4-V500 (BD Biosciences) dyes. Gating was performed on CD45-positive lymphocytes to generate these bivariate plots.

 


Figure 2. Six-color immunophenotyping analysis on the Attune® Acoustic Focusing Cytometer with red laser option.
Mouse (C57BL/6J) peripheral blood is stained with a six-color immunophenotyping panel composed of anti-mouse direct conjugates: CD3e Alexa Fluor® 647, CD19 PE-Cy®5.5, CD4 PE-Cy®7, CD8 Alexa Fluor® 488, CD11c APC-Cy®7, and NK1.1 PE direct antibody conjugates. Gating was performed on lymphocytes for each of the three bivariant plots. (A–C) A two-color representation of the six-color panel identifying the major T, B, and NK/NKT cell subsets of the lymphocyte populations. (A) Mouse T cells stained with CD3e on the x-axis and B cells stained with CD19 on the y-axis; and (B) CD4- and CD8-positive cell populations. (C) NK1.1 stains several distinct cell populations, including NK and NKT cells.

 

A No-Lyse, No-Wash Method for Immunophenotyping With Blue/Violet Laser

Immunophenotyping whole blood presents a challenge due to the limited sample volume available (≤100 μL/day/animal),1 particularly in longitudinal studies. Specifically, these small volumes limit the ability to perform multicolor phenotyping experiments with the required compensation and fluorescence-minus-one (FMO) controls.

Methods previously described using a no-lyse no-wash staining protocol do so with the loss of light scatter resolution.  Loss of cell population identification using light scatter in these applications is a limitation of hydrodynamic focusing used in traditional flow cytometry to orient cells in the flow stream. In addition, the sample dilution required in no-lyse, no-wash methods (to achieve low coincidence with red blood cells and platelets) generally dilutes the cell sample to such an extent that the time required to acquire sufficient events at the flow rates available in those instruments is inordinately long.

The acoustic focusing technology allows the Attune® cytometer aligns cells in the core stream using acoustic forces that are independent of the fluid stream. This allows a precise alignment of cells in the core and much higher throughput than is possible with traditional flow cytometry. With the Attune® Acoustic Cytometer it is now practical to process stained whole blood without the need to neither lyse red blood cells (RBCs) nor incorporate wash steps for removal of RBC fragments and platelets. This method delivers additional time savings by eliminating sample preparation steps, and eliminating lysis and wash steps to avoid sample loss.

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Multicolor Immunophenotyping Analysis

Acoustic focusing also enables both longer cell interrogation times and higher throughput, which provides higher precision analysis of dilute samples without compromising the speed of collection for multicolor immunophenotyping analysis.

Immunophenotyping Stem Cells

Immunophenotyping data using adult human mesenchymal stem cells (hMSC) were also collected on the Attune® Acoustic Focusing Cytometer.

The Attune® Acoustic Focusing Cytometer is for research use only. It is not intended for animal or human therapeutic or diagnostic use.