- Sony Biotechnology
- Sony Biotechnology
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.
|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|
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).
|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%|
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.