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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
617/30 BV605 Mouse anti-CD20
665/30 BV650 Mouse anti-CD45RA
720/60 BV711 Mouse anti-HLA-DR
785/60 BV750 Mouse anti-CD4
Table 2. MA900 instrument configuration
Sony Biotechnology Inc.
Application Data, Flexible multicolor panel design using the MA900 cell sorter
Page 2 of 4
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).
Sony Biotechnology Inc.
Application Data, Flexible multicolor panel design using the MA900 cell sorter
Page 3 of 4
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
CD20 Brilliant Violet 605
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
CD27 Brilliant Violet 510
1051041030
106
104
105
103
0
C
CD
8 P
erC
PC
y5.5
CD4 Brilliant Violet 750
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
CD4 Brilliant Violet 750
1051040
106
104
105
103
0
CD8 100%
Sony Biotechnology Inc.
Application Data, Flexible multicolor panel design using the MA900 cell sorter
Page 4 of 4
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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Table 3. Sort performance
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