flexible multicolor panel design using the ma900 cell sorter

Flexible multicolor panel design using the MA900 cell sorter

The MA900 from Sony meets the demands of most sorting applications, supporting up to 4 lasers, 12 fluorescence parameters, and 4-way sorting. Power-ful, modern technologies built into the MA900 system dramatically simplify operation to make sorting less subjec-tive and improve reliability. The optical design of the MA900 features a free-form PMT layout which expands fluorochrome choices without the need to reconfigure the system for each application (Table 1).

In this application note, we highlight the use of the MA900 cell sorter configured with 2 lasers (488 nm and 405 nm) for analysis and sorting of a 10-color immu-nophenotyping panel. Human periph-eral 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 strate-gies, and sort performance was evaluat-ed by performing 4-way sorting of CD4+ T cells, CD8+ T cells, NK cells, and B cells, followed by post-sort analysis.

Sorter A (3 laser)

488 nm

Alexa Fluor® 488

PE

PerCP-Cy5.5

PE-Texas Red®

PE-Cy7

405 nmHorizon V450

Horizon V450

638 nm

APC

Alexa Fluor 700

APC-Cy7

MA900 (2 laser)

488 nm

Alexa Fluor® 488

PE

PerCP-Cy5.5

PE/Dazzle

405 nm

BV421

BV510

BV605

BV650

BV711

BV750

Table 1. Transferring a 10-color panel from 3-laser Sorter A to the 2-laser MA900

Sony Biotechnology Inc.

Application Data, Flexible multicolor panel design using the MA900 cell sorter

1 of 4

➔

Laser Filter Fluorochrome Antibody

488 nm 525/50 Alexa Fluor® 488 Mouse anti-CD3

585/30 PE Mouse anti-CD19

617/30 PE/Dazzle Mouse anti-CD16

695/50 PerCP-Cy5.5 Mouse anti-CD8

785/60 PE-Cy7 Not used

405 nm 450/50 BV421 Mouse anti-CCR7

525/50 BV510 Mouse anti-CD27

585/30 BV570 Not used


665/30 BV650 Mouse anti-CD45RA

720/60 BV711 Mouse anti-HLA-DR


Table 2. MA900 instrument configuration



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Methods

Whole blood was lysed with RBC Lysis Buffer and stained with antibodies against Alexa Fluor® 488 CD3, PE CD19, PerCP-Cy™5.5 CD8, PE/Dazzle™ CD16, Brilliant Violet 421™ CCR7, Brilliant Violet 510™ CD27, Brilliant Violet 605™ CD20, Brilliant Violet 650™ CD45RA, Brilliant Violet 711™ HLA-DR, and Brilliant Violet 750™ CD4 (Table 2). 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 subpopulations for sorting.

B

CD

19 P

E

CD3 Alexa Fluor 488

CD3 73.93%

CD19 9.61%

1051041030

105

104

103

0

A

BSC

FSC

800600400200

800

600

400

200

0

x1,0

00

Lymphocytes 30. 81%

x1,000

Data

Stained cells were analyzed on the MA900. Lymphocytes were gated using the scatter gate (A). The CD3+ population (B) was used for gating CD4+ and CD8+ cells (C). CCR7+CD45RA+ lymphocytes are shown in (D). CD19+CD20+ B cells were gated from lymphocytes (E), and the HLADR expression of B cells was analyzed (F). CD16+ NK cells (G) were gated from lymphocytes. CD27 expression of CD45RA+ subsets of lymphocytes was analyzed (H). Using the 4-way sort mode on the MA900 cell sorter, CD4+ and CD8+ T cells, CD19+ B cells, and CD16+ NK cellswere sorted. Post-sort analysis of each sorted population is shown (I–L).



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I

CD

8 P

erC

PCy

5.5

CD4 Brilliant Violet 750

1051040

106

104

105

103

0

CD4 99.75%

L

CD

3 A

lexa

48

8

CD16 PE/Dazzle

1061051041030

105

104

103

0

CD16 98.75%

K

CD

19 P

E

CD3 Alexa Fluor 488

1051041030

105

104

103

0

CD19 99.03%

E

CD

19 P

E


1061051040

106

105

104

103

0

CD19+ CD20+ 36.56%

G

CD

3 A

lexa

Flu

or

48

8

CD16 PE/Dazzle

CD16 6.14%

1061051041030

105

104

103

0

H

CD

45R

A B

rilli

ant

Vio

let

60

5


1051041030

106

104

105

103

0

C

CD

8 P

erC

PC

y5.5


CD8+ 41.41% CD4 + CD8+ 0.44%

CD4- CD8- 4.35% CD4 53.8%

1051040

106

105

104

103

0

D

CD

45R

A B

rilli

ant

Vio

let

650

CCR7 Brilliant Violet 421

1051041030

106

105

104

103

0

A

E

I

C

G

D

H

B

F

J K L

F

Even

ts

HLA-DR Brilliant Violet 711

1061051040

55

44

33

22

11

0

HLA-DR 87.74%

J

CD

8 P

erC

PC

y5.5


1051040

106

104

105

103

0

CD8 100%



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Subset Pre Sort: % Parent Post Sort: % Parent

CD4+ T cells 40.67% 99.75%

CD8+ T cells 53.25% 100%

CD19+ B cells 9.82% 99.03%

CD16+ NK cells 3.05% 98.75%

Summary The MA900 cell sorter model (LE-MA900CP) equipped with 2 lasers, 488 nm and 405 nm, analyzed a 10-color immunophenotyping panel. Using hier-archical and boolean gating strategies, 11 distinct subsets of T, B, and NK popula-tions were identified. Of these subsets, four target populations were purified by sorting. Reanalysis of the sorted popula-tions showed that they were isolated at purities higher than 98% and at an efficiency of >80%.

Alexa Fluor® and Texas Red® are registered trademarks of Life Technologies Corporation. Cy is a trademark of GE Healthcare. PE/Dazzle is a trademark of BioLegend.

©2019 Sony Biotechnology Inc. All rights reserved. Sony and the Sony logo are trademarks of Sony Corporation. For non-clinical research use only. Not for use in diagnostic or therapeutic procedures, or for any other clinical purpose. All other trademarks are property of their respective owners. The MA900 cell sorter is classified as a Class 1 laser product. Specifications subject to change without notice. For Research Use Only.8.03.052119.0

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Europe

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Table 3. Sort performance

flexible multicolor panel design using the ma900 cell sorter

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