Bigfoot Spectral Cell Sorter Sample Data

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.

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 in key channels.

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.