MA900 instrument configuration for a 12-color immunophenotyping panel
Table 1. MA900 instrument configuration for a 12-color immunophenotyping panel

The MA900 from Sony meets the demands of most sorting applications, supporting up to 4 lasers, 12 fluorescence parameters, and 4-way sorting. Powerful, modern technologies built into the MA900 system dramatically simplify operation to make sorting less subjective and improve reliability.

For this application note, the MA900 cell sorter was used for analysis and sorting of a 12-color immunophenotyping panel (Table 1). Human peripheral blood mononuclear cells (PBMCs) were stained with reagents for analysis of common T-cell, B-cell, NK-cell, and monocyte subsets. These subsets were identified using different gating strategies, and sort performance was evaluated by performing 4-way sorting of T cells, NK cells, B cells, and monocytes, followed by post-sort analysis.



Whole blood was lysed with RBC Lysis Buffer and stained with antibodies against Alexa Fluor® 488 CD3, PerCP-Cy™5.5 CD4, Brilliant Violet 570™ CD8, APC-Cy™7 CD14, PE/Dazzle™ CD16, Brilliant Violet 421™ CD19, Brilliant Violet 605™ CD20, Brilliant Violet 510™ CD27, APC CD45RA, PE-Cy7 CD56, Alexa Fluor® 700 HLA-DR, and PE TCR gamma/delta. All reagents were from Sony Biotechnology Inc. Cells were incubated with the antibodies for 20 minutes on ice, washed twice with staining buffer, and resuspended in phosphate buffered saline (PBS) and kept on ice until further analysis.

The MA900 cell sorter was set up with a 100-μm microfluidics sorting chip using the Autocalibration feature on the system. Fluorescence compensation was done with single stained control tubes and the Cell Sorter Software V3.0 to generate the spillover matrix. Using the gating strategy described in the following section, the multicolor tube was analyzed to identify the populations of interest for sorting.



Scatter was used for gating lymphocytes (A). The CD3+ population (B) was used for gating CD4+ and CD8+ cells (C). CD4+ T-cell subsets were identified based on CD45RA and CD27 expression (D). CD16+CD56+ NK cells were gated from CD3- cells (E). HLA-DR expression of the CD19+ B cells (F) and CD20+CD3- B cells (G) was determined.

CD14+ monocytes were identified based on scatter (H). CD3+ T cells (B), CD16+CD56+ NK cells (E), CD19+HLA-DR+ B cells (F), and CD14+ monocytes (H) were sorted on a 3-laser MA900 using the 100-μm chip at 30 kHz. Post-sort analysis of each sorted population is shown in Figure 1 (I,J,K,L).


MA900 Post-sort analysis of cells
Figure 1. Post-sort analysis of cells.



MA900 Sort Performance
Table 3. MA900 sort performance

The MA900 cell sorter model (LE-MA900EP) equipped with 3 lasers, 488 nm, 405 nm, and 638 nm, analyzed a 12-color immunophenotyping panel. Using hierarchical and boolean gating strategies, 11 distinct subsets of T, B, NK, and monocyte populations were identified. Of these subsets, four target populations were purified. Reanalysis of the sorted populations showed that they were isolated at higher purities. Sort performance is shown in Table 3.



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