Performance and application data for the Bigfoot Spectral Cell Sorter

Performance data shows that the Bigfoot Spectral Cell Sorter resolves fluorescent signals accurately and precisely. Application data demonstrates the level of gating resolution and sort purity needed for studies of immunophenotyping and marker expression.

 

For additional application data, download the application notes listed in this section and on the Resources page.


8-peak bead resolution

Spherotech™ 8-peak beads run in key channels on the Bigfoot Spectral Cell Sorter show narrow, well-defined peaks.

8-peak bead resolution in key channels.

histogram for popular dyes show distinct peaks

Using the gated sort regions like the Treg gate above, six populations were sorted: CD8+ and cytotoxic T cells, B cells, NK cells, monocytes, and Tregs. For post-sort verification, each collection tube was reanalyzed for purity. Analysis of the sort populations (four of which are shown in the figure) demonstrated that the spectral unmixing capability of the Bigfoot Spectral Cell Sorter produced results with high efficiency and more than 98% purity. This sorting performance is equivalent to sorters using traditional compensation, allowing users to seamlessly transition between the two methods as experimental complexity increases.

Reanalysis of collection tubes.

Following the sort, collection tubes were reanalyzed for purity, demonstrating well-defined populations of (A) CD8 T cells (CD8strong), (B) monocytes (CD14+), (C) B cells (CD19+CD20+), and (D) cytotoxic T cells (CD8dimCD56+).

Bigfoot cell sorter produces spectrally unmixed highly pure T cells, cytotoxic T cells, B cells and monocytes

Blood cell differentiation using light scatter

Although polarization is a property of laser light that is often ignored in flow cytometry, it can be useful in differentiating certain cell types. For example, eosinophil granulocytes in human blood show relatively higher levels of depolarized laser light in a side scatter (SSC) detector due to their birefringent properties. Analysis of polarization has also been found useful in malaria diagnosis and marine phytoplankton studies.

 

All models of the Bigfoot Cell Sorter include additional FSC (front scatter) and SSC detectors for depolarized light detection. The figure shows the use of the SSC depolarized detector to identify eosinophils in lysed human whole blood. This differentiation does not require staining, leaving cells undisturbed and saving money on fluorophores. By adding a stain for CD16, other cell populations can be identified as well.

Analyzing blood cell populations using light scatter and CD16.

Lysed normal human whole blood was acquired and analyzed on a Bigfoot Spectral Cell Sorter. (A) SSC x SSC Polar dot plot shows eosinophils, with higher depolarized SSC, highlighted in red. (B) FSC x SSC contour plot shows eosinophils with high FSC and SSC signal due to their larger size compared to other cells. (C) An unstained sample shows the autofluorescence of eosinophils in the BV510 channel. (D) Dot plot of CD16 BV510 stained cells shows CD16-negative eosinophils and highlights other cell populations based on CD16 expression.

Bigfoot Cell Sorter produces pure eosinophils using light scatter/no staining

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.
For Research Use Only. Not for use in diagnostic procedures.