Performance and application data for the Bigfoot Cell Sorter

Performance data shows that the Bigfoot Cell Sorter resolves fluorescent signals accurately and precisely. Application data demonstrates the level of gating resolution and sort purity needed for immunophenotyping analysis and immune cell isolation, even for rare populations. We also demonstrate the Bigfoot Cell Sorter’s ability to sort single cells into 96 and 384 well plates with high precision and accuracy.


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

6-way immune-cell sorting with a 28-color panel

The Invitrogen Bigfoot Cell Sorter can resolve high dimensional data by unmixing the spectral signatures of overlapping dyes. This allows greater panel expansion and consequently the amount of information that can be gathered from each sample. We have demonstrated that this 28-color panel can be used to identify up to 20 different populations from one sample. From those populations, the Bigfoot instrument can sort six ways, simultaneously, with high efficiency and purity, including more rare subsets.



Gating layout for identifying and sorting mouse spleen cells using a 28-color immunophenotyping panel.

Sequential gate progression starts at the top and moves from left to right. When more than one population is identified in the same plot, arrows are used to delineate gate progression for the different subpopulations. Green shapes represent the final gating strategy for identified subsets and orange shapes represent the 6 sort targets (DCs, NKs, Tregs, T CD8+ Naïve, T1B, and MZB). Note: pDCs = plasmacytoid dendritic cells, DCs = dendritic cells, NKs = natural killer cells, Tregs = regulatory T cells, T CM = central memory T cell, T EM = effector memory T cell, MZB = marginal zone B cells, T1B = transitional 1 B cells, T2B = transitional 2 B cells, and T3B = transitional 3 B cells.

Sorting efficiency and recovered cell percentages for mouse spleen cells.

Sorting efficiency (A). Samples were acquired at a constant flow rate to maintain a speed of 2,200-2,800 events per second while sorting. Sorting priority index logic was based on the target population frequency on the spleen sample, with the less-abundant events receiving the highest priority. DCs were less abundant in the spleen sample and were therefore placed in the right-3 position with the highest priority, followed by the MZB placed in the left-3 position. The T1B were placed in right-2, CD8+ naïve in left-2, Tregs right-1, and NKs left-1.

Percentage of sorted cells that can be recovered for downstream analysis/applications (B). Recovery was assessed by acquiring the sorted samples on an Attune NxT flow cytometer, gating the sample on scatter and retrieving the cell concentration of the sorted samples (events/µL). Sample volume was obtained by subtracting the weight of the empty tubes before sort from the weight of the tubes after sort. Adjusted sorted cell numbers were obtained by multiplying cell concentration to sample volume. The number of target cells in each sorted tube was divided by the number of target cells the Bigfoot instrument showed as sorted for each tube. The result is the calculated recovery percentage for each tube.

Data are represented as mean ± standard deviation for 3 independent experiments. Note: MZB = marginal zone B cells, NKs = natural killer cells, Tregs = regulatory T cells, T1B = transitional 1 B cells, and DCs = dendritic cells.

Purity check of sorted spleen cells.

After sorting and assessing total sort numbers, the samples were analyzed on the Bigfoot instrument for purity assessment. The gates were not adjusted for the post-sort purity assessment.

Multi-way plate sorting: speed and accuracy

The Bigfoot Cell Sorter demonstrates exceptional capabilities in high-throughput plate sorting, achieving unprecedented sort speeds, precise deposition accuracy, and single-cell recovery down to small volumes. Innovative features such as 4-way sorting into 96-well and 384-well plates, built-in calibration, media detection imaging, and robust hardware set it apart from other instruments. The data presented here establish the Bigfoot Cell Sorter as a game-changing solution for researchers seeking consistent and efficient single-cell deposition into multi-well plates, enhancing workflows and paving the way for transformative discoveries.


You can read the full white paper on high-throughput plate sorting here


Test pattern with alignment targets of four-way plate sorting mode.

(A) for 96-well plates and (B) for 384-well plates.

Accurate droplet deposition.

Images show 96-well and 384-well PCR plates following multi-droplet event sorting suspended in HRP solution in a 96-well and a 384-well cell culture–treated plate. (A) In a 96-well plate, wells in rows A–D received 4, 3, 2, and 1 droplet respectively, as evidence by blue gradient from strong to pale. Wells in row E received no droplets (confirmed by no color), while rows F–H received 1 droplet per well. (B) In a 384-well plate, odd rows (starting with A) received one droplet resulting in a blue color, while even rows (starting with B) received no droplets and were colorless.

CHO cell occupancy in multiwell plates following sorting.

Representative images of CHO cell occupancy in (A) 96-well plate and (B) 384-well plate, imaged using the EVOS M7000 Imaging System. Cells were imaged using the Invitrogen EVOS Light Cube, DAPI (357/447 nm), to visualize Hoechst 33342 staining.


8-peak bead resolution

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

8-peak bead resolution in key channels.

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 (forward scatter) and SSC (side scatter) 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 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.

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.