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.

Laser	Filter	Fluorochrome	Antibody	Catalog No.
488 nm	525/50	Alexa Fluor® 488	Alexa Fluor® 488 Anti-human CD3 Antibody	2102075
	585/30	PE	PE Anti-human TCR γ/δ Antibody	2256050
	617/30	PE/Dazzle	PE/Dazzle™ 594  Anti-human CD16 Antibody	2110270
	695/50	PerCP-Cy5.5	PerCPCy5.5  Anti-human CD4 Antibody	2187140
	785/60	PE-Cy7	PE/Cy7 Anti-human CD56 Antibody	2412550
405 nm	450/50	BV421	Brilliant Violet 421™ Anti-human CD19 Antibody	2111170
	585/30	BV570	Brilliant Violet 570™ Anti-human CD8a Antibody	2105190
	617/30	BV605	Brilliant Violet 605™ Anti-human CD20 Antibody	2111670
	525/50	BV510	Brilliant Violet 510™ Anti-human CD27 Antibody	2114180
638 nm	665/30	APC	APC Anti-human CD45RA Antibody	2120560
	720/60	Alexa Fluor® 700	Alexa Fluor® 700 Anti-human HLA-DR Antibody	2235070
	785/60	APC-Cy7	APC-Cy7 Anti-human CD14 Antibody	2109100

Methods

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.

Data

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).

Summary

Subset	Pre Sort: % Parent	Post Sort: % Parent
CD3+ T cells	62.2%	99.49%
CD19/20+ B cells	31.59%	99.65%
CD16/56+ NK cells	57.33%	99.21%
CD14+ monocytes	3.1%	97.13%

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 2.