sp6800 spectral analyzer · 2017-08-02 · sp6800 spectral analyzer the sony sp6800 spectral...
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Sony Biotechnology Inc.
SP6800Spectral Analyzer
SP6800 Spectral Analyzer
The Sony SP6800 Spectral Analyzer uses spectral technology to optimize sensitivity and enhance dim signal detection by collecting photons from 420nm to 800nm. Spectral technology also simplifies multicolor panel design, by eliminating band-pass filters and conventional compensation matrices while delivering better data and simplifying visualization for the study of heterogeneous populations.
Novel global standardization mode automatically sets the system to a master specification with a single click. This capability eliminates instrument variability from day to day and across multiple instruments for greater reliability.
Advanced electronics and patented optical technologies bring simplicity to Spectral Analysis workflows. Sony’s patented FlowpointTM core stability and tracking system and automated QC ensure the highest resolution possible of target populations.
The system features easy to use software that automates alignment and laser delay with set up wizards and simplified voltage settings. Each system includes FCS ExpressTM software in addition to Sony analysis packages to offer the highest flexibility in analysis.
• Spectral analysis technology optimizes sensitivity while simplifying application design and workflow.
• Enhances dim signal detection for better visualization of rare populations, fluorescent proteins, and fluorochromes excited by multiple lasers.
• Novel global standardization mode automatically sets the system to a master specification with a single click.
• Easy to use software features include automated alignment and laser delay via set up wizards, easy acquisition, and flexible analysis using both Sony and FCS Express software included with every system.
3
The SP6800 spectral flow analyzer improves sensitivity and simplifies application design, workflow, and analysis over conventional flow cytometers. This is achieved using spectral analysis technology, advanced electronics, and patented optical technologies. These capabilities, unique to Sony Biotechnology systems, allow experienced and novice flow cytometrists to achieve greater flexibility for panel design and more accurate visualization of results.
SP6800 System Overview
Microfluidics flow cell chip maximizes signal with auto positioning to guarantee high sensitivity. Made of durable plastic with an embedded quartz cuvette, the chip is easy to replace when needed.
The 405nm, 488nm and 638nm excitation lasers are positioned to reduce fluorescent noise. They enable the system to support 16 or more fluorescent parameters.
Emitted light is directed through through a 32 Channel PMT that produces 66 data points of signal detection to analyze emitted photons from 420nm to 800 nm to ensure accurate visualization.
A unique prism collection systemDelivers light through 10 consecutive prisms allowing optimal signal separation while minimizing light loss.
The Flowpoint detection system precisely tracks the core stream shape and position in the flow cell as well as the cross sectional position of each passing particle to provide highly reliable measurements. This patented technology visualizes core stability and enables the highest resolution.
Scatter analysisForward and Side Scatter parameters to allow relative size and complexity measurements.
All events
FSC_A
SSC
_A
20
4
3
2
1
40 60x1,000
x1,0
00
4
The SP6800 software is easy to learn and use. It guides researchers from set-up to panel design, acquisition, analysis, and shutdown.
User preferences allow users and administrators set up options for overall instrument operation and experiment set up to facilitate use and unattended operation procedures.
Software
Standardization mode sets the system to a master specification to eliminate variability in a single instrument or among instruments located across sites. This unique capability allows experiments performed on any Sony cell analyzer to produce highly reliable, accurate, and reproducible results. This function also eliminates setup subjectivity for collaborative or long term studies with different operators or experience levels across sites.
Standardization
At start up Align Check and Performance QC wizards check instrument calibrations, using beads to ensure the instrument is operating optimally. On screen instructions guide the user through procedures, then display progress and report results. The performance report displays MESF, Q, and B values to describe real-time fluorescence detection performance. If desired, Align Check and Performance QC reports can be displayed in historical context.
System Start Up
When engaged, standardization mode sets the SP6800 to predetermined global settings that eliminate instrument variability.
5
On shutdown, the SP6800 software guides the user though pre –shutdown cleaning and shuts down the instrument automati-cally. Software wizards are also available to guide users through Bleach Cleaning and Rinse procedures.
All acquisition functions, including instru-ment settings are controlled from the Acquisition Window. Worksheet tools let users choose how the data is displayed- (such as plot types), and customize for their analysis needs. Plots and statistics provide real-time information during acquisition. To increase sample acquisition to the cuvette, a variable booster lets users set acquisition speed from low (33ul/min) normal or high (250ul/min) offering flexibility.
Acquisition Functions and Analysis System Shutdown
Experiments can be created using a template, an existing experiment, a single stained, or multicolor assay, in the Create Experiment window. Users can point and click to choose (or edit) an existing ex-periment and can easily select templates for wells or plates when creating a new experiment. A setup Assay Wizard guides users through the creation of a single-stained or multicolor assay simplifying experiment creation.
The Spectral Library lets users create a personal reagent library that simplifies ex-periment creation and saves time. An Acquisition Wizard assists the users with step by step instructions to acquire and analyze single positive controls for your spectral library. Once acquired the spectral reference for that reagent including the spectral index are available for future use. Information from the spectral library is avail-able to users with a simple click improving accuracy and streamline panel design.
Spectral Library Experiment Creation
6
Spectral Analyzers from Sony include FCS Express, from De Novo Software. FCS Express offers a range of new analysis tools from live gating to batchanalysis. Native support for Sony Spectral data files to enable sophisticateddata transformations and visualizations such as spectral overlays, tSNE,Spade, and heat maps.
FCS Express from De Novo Software
105
106
FOX
P3
: PE-
Daz
zle5
94 -
Are
a
CD4 : BV421 - Area
104
103
-102
102
-103
103 104 105 106
Inte
nsi
ty
Height (Wavelength)
FOXP3 +
FOXP3-
420.0 515.0 610.0 705.0 800.0
107
106
105
104
103
102
101
Inte
nsi
ty
Height (Wavelength)
FOXP3 +
FOXP3-
420.0 515.0 610.0 705.0 800.0
107
106
105
104
103
102
101
105
FOX
P3
: PE-
Daz
zle5
94
- A
rea
CD4 : BV421 - Area
104
103
102
102
-102
103 104 105 106
SSC
- A
rea
(x 1
00
0)
CD45 : BV711 - Area
Lymphs
262.1
196.6
131
65.5
102-0.1
-103 103 104 105 106
Inte
nsi
ty
Height (Wavelength)
FOXP3 +
FOXP3-
420.0 515.0 610.0 705.0 800.0
107
106
105
104
103
102
101
Dot plots and spectral plots can be set up with live gating. Live gating allows users to find positive and negative populations quickly and easily for added assurance about target populations.
Live Gating
Flexible data analysis can include integrated spreadsheets, custom calculations, charting and regression analysis.
Batch analysis lets you process any number of samples with one click. To support presentation, data can be exported directly to PowerPoint, PDF, and Excel.
Flexible Analysis Batch and Report
7
Spectral Analysis Technology
Spectral analysis technology is the foundation of the SP6800 system. Spectral flow cytometry streamlines workflow and yields better data by keeping all the light collected. In conventional systems, overlapping fluorescence is subtracted using color compensation, so less light is collected. Instead, spectral flow cytometers sum the fluorescence together and then use unmixing to mathematically separate the colors. This powerful capability also simplifies workflow including panel design, and improves visualization of autofluorescence.
A unique prism collection system delivers emitted light to a 32 channel PMT. This produces 66 data points of signal detection for fluorescence and bright auto fluorescence to achieve accurate visualizations of fluorescent populations. This lets researchers see the complete spectral fingerprintof each fluorochrome from 420nm to 800nm.
Visualization of fluorescent cell populations using the analyzer’s unique prism collection system.
A powerful capability of spectral technology is Unmixing. This allows researchers to separate fluorophores into pure signals that measure the quantity of each fluorophore at each pixel to more accurately measure data for analysis.
Spectral unmixing separates each spectral fingerprint to better visualize each fluorochrome marker. Unlike conventional filtering where overlapping signals are lost, spectral unmixing captures the photons emitted from 420nm to 800nm. In doing so it enhances dim signal detection for better visualization of rare populations, fluorescent proteins and fluorochromes excited by multiple lasers.
This also lets researchers separate the spectra of fluorophores masked by autofluorescence by extracting it from the signal and creating it into a unique fluorescent parameter. With a clear signal for each color channel unaffected by overlapping signals (spillover) and autofluorencence, spectral analysis yields better, unbiased data for analysis.
Spectral UnmixingLD
-48
8_A
106
104
103
102
101
100
105
E
Wavelengths500 600 700 800
Spectral analysis reduces false-positives and delivers more accurate analysis over conventional flow cytometry. Mouse splenocytes were stained with CD4 APC and anti-IL-2 PE. A. In this conventional density plot it is unclear if the light-blue region is a dim PE, weak double positive, or non-specific binding. B. Using Spectral analysis the spectral data of each region is compared against the Spectral Library to unmix the sample. This reveals the light blue region is high auto-fluorescence. Representative data collected on SP6800.
CD
4_A
PC
_ALD
-48
8_A
105
104
103
102
101
-101
106
104
103
102
101
100
105
E: 95.81%
G: 2.18%
F: 1.28%
CD
4_A
PC
_A
IL2_PE_A
105
104
103
102
101
-101
-102100102
E: 95.65%
G: 8.63%
F: 1.42%
103 104 105
E
Wavelengths500 600 700 800
Wavelengths Wavelengths
LD-4
88
_A
106
104
103
102
101
100
105
G
500 600 700 800
LD-4
88
_A
106
104
103
102
101
100
105
F
500 600 700 800
102 104 105-102100
IL2_PE_A103
A B
Unstained cells
HighAuto-fluorescence
PE Positive cells
Unmixing
=Spectral Unmixing separates each spectral fingerprint for complete and optimal visualization of fluorochromes.
8
H-Q2: 3.50%H-Q1: 88.02% H-Q1: 91.66%
H-Q4: 0.15%H-Q4: 0.07%
H-Q3: 8.33%H-Q3: 8.24%
All events105
104
103
102
-101
-102
-102 102
0GFP_A
Ale
xa F
luo
r 70
0_A
103 104 105
H-Q2: 0.04%
All events105
104
103
102
-101
-102
-102 102
0GFP_A
Ale
xa F
luo
r 70
0_A
103 104 105
H-Q2: 3.50%H-Q1: 88.02% H-Q1: 91.66%
H-Q4: 0.15%H-Q4: 0.07%
H-Q3: 8.33%H-Q3: 8.24%
All events105
104
103
102
-101
-102
-102 102
0GFP_A
Ale
xa F
luo
r 70
0_A
103 104 105
H-Q2: 0.04%
All events105
104
103
102
-101
-102
-102 102
0GFP_A
Ale
xa F
luo
r 70
0_A
103 104 105
Y: 0.27%
S: 0.30%
R: 95.62%
F4/80_PE_A
Wavelength
S
LD40
5/63
8_A
420
106
105
104
103
102
-101
-102
550 680 800
O106
105
104
103
102
-101
-102
-102 -101 102 103 104 105 106
B22
0_B
V65
0_A
T: 0.39%
Wavelength
V
LD40
5/63
8_A
420
106
105
104
103
102
-102
550 680 800
Wavelength
Y
LD40
5/63
8_A
420
106
105
104
103
102
-102
550 680 800
GFP Pos: 0.90%
GFP neg: 98.74%
All events106
-103
GFP_A
SSC
_A
105
104
103
102
101
100
-102 103 104 105
GFP Pos: 0.21%
GFP neg: 99.66%
All events106
-103
GFP_A
SSC
_A
105
104
103
102
101
100
-102 103 104 105
GFP Pos: 0.90%
GFP neg: 98.74%
All events106
-103
GFP_A
SSC
_A
105
104
103
102
101
100
-102 103 104 105
GFP Pos: 0.21%
GFP neg: 99.66%
All events106
-103
GFP_A
SSC
_A
105
104
103
102
101
100
-102 103 104 105
In conventional flow cytometry cellular auto-fluorescence produced by pyridine (NAD/NADH), flavin (FMN, FAD), and other intracellular oxidative reactions can cause fluorescent signal contamination of other fluorescent markers. Other common sources of auto-fluorescence include cell fixation and permeabilization. Using spectral technology, auto-fluorescent spectral fingerprints can be subtracted to allow researchers to see the true fluorescent population.
Unstained mouse splenocytes were analyzed with the SP6800 revealing three distinct auto-fluorescent populations. Using the spectral fingerprints obtained in analysis, the appropriate auto-fluorescence can be removed, increasing the precision and quality of results.
Auto-fluorescence can result in the appearance of additional cell populations leading to erroneous data interpretation. A. T cells expressing GFP and stained with an antibody conjugated to Alexa Fluor® 700 were analyzed with the SP6800. A double positive population is present in the uncorrected density plot. B. This double positive population disappears when the auto-fluorescent spectral fingerprint is subtracted. C. Liposomes from cells expressing GFP were analyzed. D. This uncovered a small positive GFP population after auto-fluorescence was subtracted.
Subtracting Auto-Fluorescence Improves Visualization
A. With Auto-fluorescence
C. With Auto-fluorescence
B. Without Auto-fluorescence
Activated T Cells with GFP and Alexa Fluor 700
Liposomes with GFP
D. Without Auto-fluorescence
A correction system adjusts the offset and sensitivity of each channel of the 32 channel PMT to ensure a uniform and accurate measurement of fluorescent emission from 420nm to 800nm. The corrective system brings a standardization to all 32 PMTs ensuring the user is getting the most reliable data with the ease of adjusting only one voltage. This saves time over conventional flow cytometry operation where users must calibrate each individual PMT.
Uniform measurement of Fluorescent Emissions
Pre and Post Correction Profile. Each graph illustrates pre and post correction in the SP6800 32 channel PMT. The correction improves accuracy of spectral visualization.
Spectral emission of PE Cy5 pre and post correction. Corrections support accurate unmixing of closely overlapping fluorescence spectra.
Post-correction120
100
80
60
40
20
01 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
Pre-correction120
100
80
60
40
20
01 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
Pre correction Post correction
PE-Cy5 PE-Cy5
All events
Channel
LD4
88
_H
106
105
104
103
102
101
100
1 10 20 32
All events
Channel
LD4
88
_H
106
105
104
103
102
101
100
1 10 20 32
Correction
9
500 600Wavelength (nm)
700 800
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
• Alexa Fluor® 488; 488nm • Alexa Fluor 532; 488nm • PE; 488nm • PE DazzleTM 594; 488nm • PE Alexa Fluor 610; 488nm • PE Cy5TM; 488nm • PerCP Cy5.5; 488nm • PE Cy7; 488nm
• BrilliantTM Blue 515; 488nm • Alexa Fluor 514; 488nm • PE Texas Red®; 488nm • PerCP eFluor 710; 488nm • PE Cy5.5; 488nm • PE Vio®770; 488nm
• FITC; 488nm • PerCP; 488nm
• PE Alexa Fluor 700; 488nmLaser
• 488nm
wavelength (nm)500 600
wavelength (nm)400 600500
Group 9 Group 10 Group 11 Group 12 Group 13 Group 14 Group 15 Group 16
• Brilliant VioletTM 421; 405nm • Pacific BlueTM; 405nm • Brilliant Violet 510TM; 405nm • Brilliant Violet 570TM; 405nm • Brilliant Violet 605TM; 405nm • Brilliant Violet 650TM; 405nm • Brilliant Violet 711TM; 405nm • Brilliant Violet 785TM; 405nm
• Alexa Fluor 405; 405nm • HorizonTM V450; 405nm • AmCyan; 405nm • Pacific OrangeTM; 405nm • Qdot® 605; 405nm • Qdot 655; 405nm • Qdot 705; 405nm • Qdot 800; 405nm
• Horizon V500; 405nm • eVolveTM 605; 405nm • eFluor 660; 405nm • Alexa Fluor 700; 638nm • APC Cy7; 638nm
• Krome OrangeTM; 405nm • Alexa Fluor 647; 638nm • APC Cy5.5; 638nm • APC Alexa 750; 638nm
• APC; 638nm • APC eFluor 780; 638nm
• Cy5; 638nm
400 500 600
Wavelength (nm)
700 800
Laser
• 405nm
• 638nm
Application data dye options and combinations. Select one from each group.
Fluorochrome Panel Guide
10
500 600Wavelength (nm)
700 800
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
• Alexa Fluor® 488; 488nm • Alexa Fluor 532; 488nm • PE; 488nm • PE DazzleTM 594; 488nm • PE Alexa Fluor 610; 488nm • PE Cy5TM; 488nm • PerCP Cy5.5; 488nm • PE Cy7; 488nm
• BrilliantTM Blue 515; 488nm • Alexa Fluor 514; 488nm • PE Texas Red®; 488nm • PerCP eFluor 710; 488nm • PE Cy5.5; 488nm • PE Vio®770; 488nm
• FITC; 488nm • PerCP; 488nm
• PE Alexa Fluor 700; 488nm
700 800
700 800
Group 9 Group 10 Group 11 Group 12 Group 13 Group 14 Group 15 Group 16
• Brilliant VioletTM 421; 405nm • Pacific BlueTM; 405nm • Brilliant Violet 510TM; 405nm • Brilliant Violet 570TM; 405nm • Brilliant Violet 605TM; 405nm • Brilliant Violet 650TM; 405nm • Brilliant Violet 711TM; 405nm • Brilliant Violet 785TM; 405nm
• Alexa Fluor 405; 405nm • HorizonTM V450; 405nm • AmCyan; 405nm • Pacific OrangeTM; 405nm • Qdot® 605; 405nm • Qdot 655; 405nm • Qdot 705; 405nm • Qdot 800; 405nm
• Horizon V500; 405nm • eVolveTM 605; 405nm • eFluor 660; 405nm • Alexa Fluor 700; 638nm • APC Cy7; 638nm
• Krome OrangeTM; 405nm • Alexa Fluor 647; 638nm • APC Cy5.5; 638nm • APC Alexa 750; 638nm
• APC; 638nm • APC eFluor 780; 638nm
• Cy5; 638nm
400 500 600
Wavelength (nm)
700 800
11
WLSM
Granulocytes
Lymphocytes
Monocytes
All events
x1,0
00
SSC
_A
CD45_PE-Cy7_A
5
4
3
2
1
-102-10
210
210
310
410
5
WLSM
CD24-CD13
Granulocytes
CD
13_P
erC
P e
flo
ur7
10_A
CD24_PE-AF610_A
104
-102
10110
210
310
410
5
103
102
-101
-102
WLSM
CD35-CD3
Natural Killer Cells
CD35-CD3 12.27%
Lymphocytes
CD
35_B
V4
21_A
CD3_Alexa Fluor 458_A
106
-102
100
102
103
105
104
103
-102
WLSM
CD35-CD3
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
110
210
6
104
103
-102
103
104
105
CD16-CD3: 10.73%
WLSM
Lymphocytes
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
010
2
104
103
-102
103
CD11c-CD14-
WLSM
CD45+
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
2
104
103
-102
-103
-103
106
105
104
CD14 positive
WLSM
Monocytes
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-101
-102
-103
105
104
Cytotoxic T Cells
WLSM
Lymphocytes
CD
8_P
E_A
CD3_Alexa Fluor 453_A-10
210
3
104
103
102
-102
-10010
2
Naive Cytotoxic T Cells
E�ector Cytotoxic T Cells E�ector Cytotoxic T Cells
WLSM
Cytotoxic T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
104
103
102
-101
-102
-103
102
101
Helper T Cells
WLSM
Lymphocytes
CD
4_P
EDaz
zle
594
_A
CD3_Alexa Fluor 453_A-10
210
3
103
-101
-102
-10010
2
Naive Helper T Cells
WLSM
Helper T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
103
102
-102
-103
-101
-10010
2
CD13-CD14
WLSM
CD4
CD
14_B
V78
5_A
CD13_PerCP eflour710_A-10
210
3
103
102
-101
-102
104
CD3-CD33
WLSM
All events
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-102
-103
105
104
PLASMA B Cells
WLSM
CD19-CD20
CD
35_B
V4
21_A
HLA-DR_PacificBlue_A-10
210
3
104
103
102
-102
-101
104
104
104
CD19c-CD20
WLSM
CD11c-CD14-
CD
23_P
E-C
y5.5
_A
CD19_PE-Cy5_A-10
210
3
104
103
-102
104
Memory B Cells
Naive B Cells
WLSM
PLASMA B Cells
CD
11b
_BV
650
_A
CD45RA_BV510_A10
210
3
104
105
103
102
-102
-101
-103
101
100
104
105
106
WLSM
Granulocytes
Lymphocytes
Monocytes
All events
x1,0
00
SSC
_A
CD45_PE-Cy7_A
5
4
3
2
1
-102-10
210
210
310
410
5
WLSM
CD24-CD13
Granulocytes
CD
13_P
erC
P e
flo
ur7
10_A
CD24_PE-AF610_A
104
-102
10110
210
310
410
5
103
102
-101
-102
WLSM
CD35-CD3
Natural Killer Cells
CD35-CD3 12.27%
Lymphocytes
CD
35_B
V4
21_A
CD3_Alexa Fluor 458_A
106
-102
100
102
103
105
104
103
-102
WLSM
CD35-CD3
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
110
210
6
104
103
-102
103
104
105
CD16-CD3: 10.73%
WLSM
Lymphocytes
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
010
2
104
103
-102
103
CD11c-CD14-
WLSM
CD45+
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
2
104
103
-102
-103
-103
106
105
104
CD14 positive
WLSM
Monocytes
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-101
-102
-103
105
104
Cytotoxic T Cells
WLSM
Lymphocytes
CD
8_P
E_A
CD3_Alexa Fluor 453_A-10
210
3
104
103
102
-102
-10010
2
Naive Cytotoxic T Cells
E�ector Cytotoxic T Cells E�ector Cytotoxic T Cells
WLSM
Cytotoxic T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
104
103
102
-101
-102
-103
102
101
Helper T Cells
WLSM
Lymphocytes
CD
4_P
EDaz
zle
594
_A
CD3_Alexa Fluor 453_A-10
210
3
103
-101
-102
-10010
2
Naive Helper T Cells
WLSM
Helper T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
103
102
-102
-103
-101
-10010
2
CD13-CD14
WLSM
CD4
CD
14_B
V78
5_A
CD13_PerCP eflour710_A-10
210
3
103
102
-101
-102
104
CD3-CD33
WLSM
All events
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-102
-103
105
104
PLASMA B Cells
WLSM
CD19-CD20
CD
35_B
V4
21_A
HLA-DR_PacificBlue_A-10
210
3
104
103
102
-102
-101
104
104
104
CD19c-CD20
WLSM
CD11c-CD14-
CD
23_P
E-C
y5.5
_A
CD19_PE-Cy5_A-10
210
3
104
103
-102
104
Memory B Cells
Naive B Cells
WLSM
PLASMA B Cells
CD
11b
_BV
650
_A
CD45RA_BV510_A10
210
3
104
105
103
102
-102
-101
-103
101
100
104
105
106
WLSM
Granulocytes
Lymphocytes
Monocytes
All events
x1,0
00
SSC
_A
CD45_PE-Cy7_A
5
4
3
2
1
-102-10
210
210
310
410
5
WLSM
CD24-CD13
Granulocytes
CD
13_P
erC
P e
flo
ur7
10_A
CD24_PE-AF610_A
104
-102
10110
210
310
410
5
103
102
-101
-102
WLSM
CD35-CD3
Natural Killer Cells
CD35-CD3 12.27%
Lymphocytes
CD
35_B
V4
21_A
CD3_Alexa Fluor 458_A
106
-102
100
102
103
105
104
103
-102
WLSM
CD35-CD3
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
110
210
6
104
103
-102
103
104
105
CD16-CD3: 10.73%
WLSM
Lymphocytes
CD
16_B
V60
5_A
CD3_Alexa Fluor 458_A-10
2-10
010
2
104
103
-102
103
CD11c-CD14-
WLSM
CD45+
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
2
104
103
-102
-103
-103
106
105
104
CD14 positive
WLSM
Monocytes
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-101
-102
-103
105
104
Cytotoxic T Cells
WLSM
Lymphocytes
CD
8_P
E_A
CD3_Alexa Fluor 453_A-10
210
3
104
103
102
-102
-10010
2
Naive Cytotoxic T Cells
E�ector Cytotoxic T Cells E�ector Cytotoxic T Cells
WLSM
Cytotoxic T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
104
103
102
-101
-102
-103
102
101
Helper T Cells
WLSM
Lymphocytes
CD
4_P
EDaz
zle
594
_A
CD3_Alexa Fluor 453_A-10
210
3
103
-101
-102
-10010
2
Naive Helper T Cells
WLSM
Helper T Cells
CD
45R
A_B
V51
0_A
CD35_BV421_A-10
210
3
103
102
-102
-103
-101
-10010
2
CD13-CD14
WLSM
CD4
CD
14_B
V78
5_A
CD13_PerCP eflour710_A-10
210
3
103
102
-101
-102
104
CD3-CD33
WLSM
All events
CD
14_B
V78
5_A
CD11c_BV711_A-10
210
3
104
103
102
-102
-103
105
104
PLASMA B Cells
WLSM
CD19-CD20
CD
35_B
V4
21_A
HLA-DR_PacificBlue_A-10
210
3
104
103
102
-102
-101
104
104
104
CD19c-CD20
WLSM
CD11c-CD14-
CD
23_P
E-C
y5.5
_A
CD19_PE-Cy5_A-10
210
3
104
103
-102
104
Memory B Cells
Naive B Cells
WLSM
PLASMA B Cells
CD
11b
_BV
650
_A
CD45RA_BV510_A10
210
3
104
105
103
102
-102
-101
-103
101
100
104
105
106
Application data using 2 lasers
16 color panel on Human Peripheral Blood
488nm Laser 405nm Laser
Examples of the resolution of several important cell populations using only two lasers with the SP6800. Clear population resolution is obtained with highly overlapping fluorochomes such as PE-Cy5/PE-Cy5.5 (G) and Pacific Blue/BV421 (H).
A
F
J
B
G
K
C
H
L
D
I
E
HLA-DR Pacific Blue
CD38 BV421
CD45RA BV510
CD33 BV570
CD16 BV605
CD11b BV650
CD11c BV711
CD14 BV785
CD3 Alexa Fluor 488
CD8 PE
CD4 PE Dazzle 594
CD24 PE Alexa Fluor 610
CD19 PE Cy5
CD20 PE Cy5.5
CD13 PerCP efluor® 710
CD45 PE Cy7
2 Lasers, 488nm Laser
2 Lasers, 405nm Laser
400 800700600
Wavelength (nm)
500
400 800700600
Wavelength (nm)
500
2 Lasers, 488nm Laser
2 Lasers, 405nm Laser
400 800700600
Wavelength (nm)
500
400 800700600
Wavelength (nm)
500
Sample Data
12
WLSM
Granulocytes
CD13+CD24+ Grans
Lymphocytes
Monocytes
All events
x1,0
00
SSC
_A
CD
13_P
erC
P e
flo
ur7
10_A
CD45_PE-Cy7_A CD24_PE-AF620_A
4
2
-102
-101
102
103
104
105
106
WLSM
Plasma B Cells: 99.57%
CD19+CD20+
CD
38_A
PC
_A
HLA-DR_PacificBlue_A-10
2-10
110
210
310
410
5
105
104
103
102
-101
-102
-103
106
WLSM
Helper T Cells: 50.38%
Lymphocytes
CD
4_P
EDaz
zle
594
_A
CD3_Alexa Fluor 488_A-10
2-10
110
210
3
101
-100
-101
WLSM
Helper Naive T Cells
Lymphocytes
CD
45R
A_A
PC-
Cy7
_A
CD38_APC_A-10
210
210
3
103
102
104
-102
WLSM
Cytotoxic T Cells
Lymphocytes
CD
8_P
E_A
CD3_Alexa Fluor 488_A-10
2-10
110
210
310
4
104
103
102
-102
WLSM
Cytotoxic Naive T Cells
Cytotoxic E�ector T Cells
Helper E�ector T Cells
WLSM
CD5+CD24+ B Cells
Lymphocytes
CD
24_P
E-A
F610
_A
CD5_BV421_A-10
210
310
5
104
103
105
106
-102
-103
-103
104
WLSM
CD13+CD33+ Monocytes
CD
33_B
V57
0_A
CD13_PerCP eflour710_A-10
210
3
102
101
103
-102
-102
Cytotoxic T Cells
CD
45R
A_A
PC-
Cy7
_A
CD38_APC_A-10
210
210
310
4
104
103
102
-102
WLSM
CD16 positive
Monocytes
CD
16_B
V60
5_A
CD3_Alexa Fluor 488_A-10
210
210
310
4
Monocytes
WLSM
CD24+CD38- Granulocytes
CD
38_A
PC
_A
CD24_PE-AF610_A-10
210
3
104
103
106
105
-102
-103
-101
-103
102
104
104
103
-102
-100
WLSM
Granulocytes
103
-102-10
110
210
310
4
102
-101
-102
-103
-103
NK Cells
CD
16_B
V60
5_A
CD3_Alexa Fluor 488_A
WLSM
Lymphocytes
104
-102-10
110
210
3
103
102
-102
CD19+CD20+C
D20
_PE-
Cy5
.5_A
CD19_PE-Cy5_A
WLSM
CD45+
104
-103
-102
103
104
105
103
-102
-103
Granulocytes
Application data using 3 lasers
16 color stained sample of Human Peripheral Blood
488nm Laser
638nm Laser
405nm Laser
Examples of the detection of several important cell populations using sixteen fluorochromes excited by three lasers. Unlike conventional flow cytometers that can detect up to five off the blue, in this example we demonstrate that with the SP6800 eight fluorochromes can be excited off the blue with clear sample resolution. Even highly overlapping fluorchromes such as PE-Cy5 and PE-Cy5.5 can be resolved.
CD3 Alexa Fluor 488
CD8 PE
CD4 PE Dazzle 594
CD24 PE Alexa Fluor 610
CD19 PE Cy5
CD20 PE Cy5.5
CD13 PerCP efluor 710
CD45 PE Cy7
HLA-DR Pacific Blue
CD5 BV421
CD56 BV510
CD33 BV570
CD16 BV605
CD38 APC
CD11c Alexa Fluor 700
CD45RA APC-Cy7
3 Lasers, 488nm Laser
400 800700600
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
500
400 800700600500
400 800700600500
3 Lasers, 405nm Laser
3 Lasers, 638nm Laser
3 Lasers, 488nm Laser
400 800700600
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
500
400 800700600500
400 800700600500
3 Lasers, 405nm Laser
3 Lasers, 638nm Laser
3 Lasers, 488nm Laser
400 800700600
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
500
400 800700600500
400 800700600500
3 Lasers, 405nm Laser
3 Lasers, 638nm Laser
13
Spectral Analysis allows multi-laser excited fluorochromes to be run without using a complicated compensation matrix.
Application Data: Brilliant Violet Dyes
Wavelength (nm)
400 800700600500
Example of Human Peripheral Blood Leukocytes was analyzed on the SP6800 and a BD LSRFortessa conventional flow cytometer. Spectral analysis was able to remove the autofluorescenece and use unmixing to separate each fluorochrome which resulted in clarity and resolution of each population in these density plots.
Sample data from Human PBMCs stained with seven markers conjugated to Brilliant Violet dyes offered by Sony Biotechnology. D. All populations were clearly resolved including fluorochomes with significant spectral overlap such as BV605 and BV650 (D).
12-color Staining of Human Peripheral Blood Leukocytes
BV421
BV510
BV570
BV605
BV650
BV711
BV785
BA C D E
CD45+
SSC
_A
CD
8_B
V57
0_A
CD45_BV711_H CD3_BV421_A
H
x1,0
00
10
8
6
4
2
102 104 105103
CD45+
102
104
103
102
-101
-102
-102 103 104 105
CD
4_B
V51
0_A
CD3_BV421_A
CD45+
102
103
102
-101
-102
-102 103 104 105C
D16
/56_
BV
605_
ACD19_BV650_A
CD45+
102
103
102
-102
-102 -101 103
CD
3_B
V4
21_A
CD14_BV785_A
CD45+
102
105
104
103
-102
102
-102-100 103
CD45+
SSC
_A
CD
8_B
V57
0_A
CD45_BV711_H CD3_BV421_A
H
x1,0
00
10
8
6
4
2
102 104 105103
CD45+
102
104
103
102
-101
-102
-102 103 104 105
CD
4_B
V51
0_A
CD3_BV421_A
CD45+
102
103
102
-101
-102
-102 103 104 105C
D16
/56_
BV
605_
A
CD19_BV650_A
CD45+
102
103
102
-102
-102 -101 103
CD
3_B
V4
21_A
CD14_BV785_A
CD45+
102
105
104
103
-102
102
-102-100 103
CD45+
SSC
_A
CD
8_B
V57
0_A
CD45_BV711_H CD3_BV421_A
H
x1,0
00
10
8
6
4
2
102 104 105103
CD45+
102
104
103
102
-101
-102
-102 103 104 105
CD
4_B
V51
0_A
CD3_BV421_A
CD45+
102
103
102
-101
-102
-102 103 104 105C
D16
/56_
BV
605_
A
CD19_BV650_A
CD45+
102
103
102
-102
-102 -101 103
CD
3_B
V4
21_A
CD14_BV785_A
CD45+
102
105
104
103
-102
102
-102-100 103
CD45+
SSC
_A
CD
8_B
V57
0_A
CD45_BV711_H CD3_BV421_A
H
x1,0
00
10
8
6
4
2
102 104 105103
CD45+
102
104
103
102
-101
-102
-102 103 104 105
CD
4_B
V51
0_A
CD3_BV421_A
CD45+
102
103
102
-101
-102
-102 103 104 105C
D16
/56_
BV
605_
ACD19_BV650_A
CD45+
102
103
102
-102
-102 -101 103
CD
3_B
V4
21_A
CD14_BV785_A
CD45+
102
105
104
103
-102
102
-102-100 103
CD45+
SSC
_A
CD
8_B
V57
0_A
CD45_BV711_H CD3_BV421_A
H
x1,0
00
10
8
6
4
2
102 104 105103
CD45+
102
104
103
102
-101
-102
-102 103 104 105
CD
4_B
V51
0_A
CD3_BV421_A
CD45+
102
103
102
-101
-102
-102 103 104 105C
D16
/56_
BV
605_
ACD19_BV650_A
CD45+
102
103
102
-102
-102 -101 103
CD
3_B
V4
21_A
CD14_BV785_A
CD45+
102
105
104
103
-102
102
-102-100 103
Wavelength
All events
LD4
88
_H
700 800600500102
103
104
105
106
Wavelength
All events
LD40
5/6
38_H
680 800550420102
103
104
105
106
CD45RA
Marker Fluorochrome 488nm Laser 405/638nm Laser
TCR-gd
CD56
CD45
CD3
CCR7
CD27
CD33
CD4
CD19
CD8
HLADR
Alexa Fluor 488
PE
PE-Dazzle594
PerCP-Cy5.5
PE-Cy7
BV421
BV510
BV570
BV605
APC
Alexa Fluor 700
APC-Cy7
Sample Data
14
Sony SP6800 Spectral Analyzer
CD4: BV605 - Area CD4: BV605 - Area CD4: BV605 - Area CD4: BV605 - Area
CD
27: B
V51
0 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
-10
3
CD
19: A
PC
- A
rea
106
105
104
0-1
04
103 104 105 106-103
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 105 106-103
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
-10
3
104103 105 106-103
103
CD4-BV605 CD4-BV605 CD4-BV605 CD4-BV605
CD
27-B
V51
010
510
410
310
2
0 103 104 105102-102
0
CD
19-A
PC
105
102
-10
2
105-102
104
103
1020 103 104
TCR
GD
-PE
105
104
103
102
-10
2
0 103 104 105-103 102
CD
56-P
E D
azzl
e10
510
410
3
102 103 104 105-102
102
-10
2
0
Sony SP6800 Spectral Analyzer
CD3: PE-Cy7 - Area
CD
45R
A: A
lexa
Flu
or
48
8 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
CD3: PE-Cy7 - Area
CCR
7: B
V4
21 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD8: Alexa Fluor 700 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
103
-10
3
0 104 105 106-104
CD3: PE-Cy7 - Area
HLA
DR
: AP
C-C
y7 -
Are
a10
610
510
4-1
03
0 104 105 106-104
103
CD3-PECy7
CD
45R
A-A
F48
810
510
410
310
2
0 103 104 105102-102
CD3-PECy7
CCR
7-B
V4
2110
510
20
-10
2
103 105-102
104
103
1020 103 104
CD8-AF700
CD
56-P
E D
azzl
e10
510
410
310
2-1
02
0 103 104 105-103
CD19-APC
HLA
-DR
-AP
CCy7
105
104
103
102 103 104 105-102
102
-10
20
Sony SP6800 Spectral Analyzer
CD3: PE-Cy7 - Area
CD
45R
A: A
lexa
Flu
or
48
8 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
CD3: PE-Cy7 - Area
CCR
7: B
V4
21 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD8: Alexa Fluor 700 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
103
-10
3
0 104 105 106-104
CD3: PE-Cy7 - Area
HLA
DR
: AP
C-C
y7 -
Are
a10
610
510
4-1
03
0 104 105 106-104
103
CD3-PECy7
CD
45R
A-A
F48
810
510
410
310
2
0 103 104 105102-102
CD3-PECy7
CCR
7-B
V4
2110
510
20
-10
2
103 105-102
104
103
1020 103 104
CD8-AF700
CD
56-P
E D
azzl
e10
510
410
310
2-1
02
0 103 104 105-103
CD19-APC
HLA
-DR
-AP
CCy7
105
104
103
102 103 104 105-102
102
-10
20
Sony SP6800 Spectral Analyzer
CD45: PerCP-Cy5.5 - Area
SSC
- A
rea
(x 1
0)
250
020
00
150
010
00
500
0
0 103 104 105 106-103
Conventional Flow Cytometer
CD3: PE-Cy7 - Area
CD
4: B
V60
5 -
Are
a10
610
510
410
3-1
03
0
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
8: A
lexa
Flu
or
700
- A
rea
106
105
104
0-1
04
0
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
0-1
03
0
103 104 105 106-103
103
CD45-PerCPCy5.5
SSC-
A(x
10
)30
00
250
020
00
100
015
00
500
0
0 103 104 105102-102
CD3-PECy7
CD
4-B
V60
510
510
20
-10
2
103 105-102
104
103
1020 103 104
CD3-PECy7
CD
8-A
F70
010
510
410
30
-10
3
1020 103 104 105-102
CD3-PECy7
CD
56-P
E D
azzl
e10
510
410
3-1
02
1020 103 104 105-102
102
Sony SP6800 Spectral Analyzer
CD45: PerCP-Cy5.5 - Area
SSC
- A
rea
(x 1
0)
250
020
00
150
010
00
500
0
0 103 104 105 106-103
Conventional Flow Cytometer
CD3: PE-Cy7 - Area
CD
4: B
V60
5 -
Are
a10
610
510
410
3-1
03
0
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
8: A
lexa
Flu
or
700
- A
rea
106
105
104
0-1
04
0
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
0-1
03
0
103 104 105 106-103
103
CD45-PerCPCy5.5
SSC-
A(x
10
)30
00
250
020
00
100
015
00
500
0
0 103 104 105102-102
CD3-PECy7
CD
4-B
V60
510
510
20
-10
2
103 105-102
104
103
1020 103 104
CD3-PECy7
CD
8-A
F70
010
510
410
30
-10
3
1020 103 104 105-102
CD3-PECy7
CD
56-P
E D
azzl
e10
510
410
3-1
02
1020 103 104 105-102
102
1
3
5
2
4
Sony SP6800 Spectral Analyzer
Sony SP6800 Spectral Analyzer
Sony SP6800 Spectral Analyzer
Sony SP6800 Spectral Analyzer
Sony SP6800 Spectral Analyzer
Conventional Flow Cytometer
Conventional Flow Cytometer
Conventional Flow CytometerConventional Flow Cytometer
Conventional Flow Cytometer
Sony SP6800 Spectral Analyzer
CD3: PE-Cy7 - Area
CD
19: A
PC
- A
rea
106
105
104
-10
40
103 104 105 106-103
Conventional Flow Cytometer
CD3: PE-Cy7 - Area
HLA
DR
: AP
C-C
y7 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD3: PE-Cy7 - Area
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
27: B
V51
0 -
Are
a10
610
510
4-1
03
103 104 105 106-103
103
0
CD3-PECy7
CD
19-A
PC
105
104
103
102
-10
20
0 103 104 105102-102
CD3-PECy7
HLA
-DR
-AP
CCy7
105
102
0-1
02
103 105-102
104
103
1020 103 104
CD3-PECy7
TCR
GD
-PE
105
104
103
102
-10
3
1020 103 104 105-102
CD3-PECy7
CD
27-B
V51
010
510
410
3
1020 103 104 105-102
102
0
Sony SP6800 Spectral Analyzer
CD3: PE-Cy7 - Area
CD
19: A
PC
- A
rea
106
105
104
-10
40
103 104 105 106-103
Conventional Flow Cytometer
CD3: PE-Cy7 - Area
HLA
DR
: AP
C-C
y7 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD3: PE-Cy7 - Area
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 104 105 106-103
CD3: PE-Cy7 - Area
CD
27: B
V51
0 -
Are
a10
610
510
4-1
03
103 104 105 106-103
103
0
CD3-PECy7
CD
19-A
PC
105
104
103
102
-10
20
0 103 104 105102-102
CD3-PECy7
HLA
-DR
-AP
CCy7
105
102
0-1
02
103 105-102
104
103
1020 103 104
CD3-PECy7
TCR
GD
-PE
105
104
103
102
-10
3
1020 103 104 105-102
CD3-PECy7
CD
27-B
V51
010
510
410
3
1020 103 104 105-102
102
0
Sony SP6800 Spectral Analyzer
CD33: BV570 - Area
CD
27: B
V51
0 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
-10
3
CD33: BV570 - Area
CD
4: B
V60
5 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD33: BV570 - Area
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 105 106-103
CD33: BV570 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
-10
3
104103 105 106-103
103
CD33-BV570
CD
27-B
V51
010
510
410
310
2
0 103 104 105102-102
0
CD33-BV570
CD
4-B
V60
510
510
20
-10
2
105-102
104
103
1020 103 104
CD33-BV570
TCR
GD
-PE
105
104
103
102
-10
2
0 103 104 105-103 102
CD33-BV570
CD
56-P
E D
azzl
e10
510
410
3
102 103 104 105-102
102
-10
2
0
Sony SP6800 Spectral Analyzer
CD33: BV570 - Area
CD
27: B
V51
0 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
-10
3
CD33: BV570 - Area
CD
4: B
V60
5 -
Are
a10
610
510
410
3-1
03
103 104 105 106-103
CD33: BV570 - Area
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 105 106-103
CD33: BV570 - Area
CD
56: P
E-D
azzl
e594
- A
rea
106
105
104
-10
3
104103 105 106-103
103
CD33-BV570
CD
27-B
V51
010
510
410
310
2
0 103 104 105102-102
0
CD33-BV570
CD
4-B
V60
510
510
20
-10
2
105-102
104
103
1020 103 104
CD33-BV570
TCR
GD
-PE
105
104
103
102
-10
2
0 103 104 105-103 102
CD33-BV570
CD
56-P
E D
azzl
e10
510
410
3
102 103 104 105-102
102
-10
2
0
Sony SP6800 Spectral Analyzer
CD4: BV605 - Area CD4: BV605 - Area CD4: BV605 - Area CD4: BV605 - Area
CD
27: B
V51
0 -
Are
a10
610
510
40
103 104 105 106-103
Conventional Flow Cytometer
103
-10
3
CD
19: A
PC
- A
rea
106
105
104
0-1
04
103 104 105 106-103
TCR
-gd
: PE
- A
rea
106
105
104
103
-10
3
103 105 106-103
CD
56: P
E-D
azzl
e59
4 -
Are
a10
610
510
4-1
03
104103 105 106-103
103
CD4-BV605 CD4-BV605 CD4-BV605 CD4-BV605
CD
27-B
V51
010
510
410
310
2
0 103 104 105102-102
0
CD
19-A
PC
105
102
-10
2
105-102
104
103
1020 103 104
TCR
GD
-PE
105
104
103
102
-10
2
0 103 104 105-103 102
CD
56-P
E D
azzl
e10
510
410
3
102 103 104 105-102
102
-10
2
0
15
F106
105
104
103
102
-101
-102
-102 -101 102 103
PE-Cy7_A
PE_
A
104 105 106
Cells
CD4_PacificBlue_A
CD
8_B
V4
21_A
-102 102
105
104
103
102
-1020
103
WLSM
A
GFP_A
-102
103
103
-102
-103
104
105
106
104
105
YFP
_A
Wavelength
800700600500
C
100
101
102
103
104
105
106
LD4
88
_A
Wavelength
800700600500
C
100
101
102
103
104
105
106
LD4
88
_A
Wavelength
800700600500
C
100
101
102
103
104
105
106
LD4
88
_A
Wavelength
800700600500
C
100
101
102
103
104
105
106
LD4
88
_A
WLSM
All events
Wavelength
LD48
8_H
800700600500
106
105
104
103
102
101
100
Common problems in flow cytometry occur when running fluorochromes with emission peaks that are too close to one another, multi-laser excitations, fluorescent proteins, and unstable tandems. The SP6800 is capable of analyzing all of these by looking at all photons from 420nm to 800nm and unmixing each unique spectral fingerprint.
Application Data: Dyes with issues
Example 1: Pacific Blue (457nm emission) and Brilliant Violet 421 (422nm emission)
Example 2: PE Cy5 (excited with 488nm and 638nm lasers) and APC (excited with 638nm laser)
Example 3: Fluorescent GFP and YFP need no special bandpass filter set with the SP6800
Example 4: Spectral Analysis unmixes the spectral fingerprint the spectral fingerprint of PE Cy7 tandem to produce excellent resolutions.
APC excitation
APC emission
PE Cy5 excitation
PE Cy5 emission
Normal PE Cy7
PE Cy7 falling apart
Unmixing
Wavelength (nm)
400 800700600500
Wavelength (nm)
400 800700600500
CD4_APC_A
-102 -101 102 103 104
106E
105
104
103
102
-101
-102
105
CD
8_P
E-C
y5_A
Sample Data
* Except for LE-SP6800ZC (This HPCV value is calculated for red laser. )
Configuration 2 laser model, 488/638 2 laser model, 405/488 3 laser model,405/488/638
Model Number LE-SP6800ZB LE-SP6800ZC LE-SP6800ZE
Optics
Laser specification
LasersSemiconductor 2 laser model488nm, 638nm
Semiconductor 2 laser model 405/488
Semiconductor 3 laser model, 405/488/638
Maximum power (at flow cell)488nm 40mW638nm 60mW
405nm 60mW488nm 40mW
405nm 60mW, 488nm 40mW 638nm 60mW
Irradiation form 2-spot dual-axis irradiation
Detection optics
Scatter signals Forward scatter (FSC), Side scatter (SSC)
Spectroscopic method Prism array spectroscopy
Fluorescent channels32 channel PMT(wavelength: 500-800nm)
32 channel PMT (wavelength: 500-800nm) PMT x 2 (420-440nm, 450-470nm)
System
Signal resolution Height 20 bit, Area 32 bit Sampling frequency: 50MHz
Measurement parameters64 channels, FSC, SSC, cell position XY
66 channels, FSC, SSC, cell position XY
Pulse shape parameters Height, Area, Width
Optical alignment Automated alignment with CorefinderTM technology
Event Rate 10,000eps (standard)/20,000eps (maximum)
Fluidics
Sample loader type Single auto-loader
Supporting sample tube 5ml polystyrene round tubes
Sheath Flow Speed Low (approx. 3m/s), Mid (approx. 5m/s), High (approx. 10m/s)
Detection channel dimensions 200μm x 200μm
Performance
Fluorescence sensitivity FITC: 120 MESF / PE: 70 MESF
Linearity FITC R2 ≧ 0.995 / PE R2 ≧ 0.995
Detection size range 0.5~40µm (beads)
Fluorescence detection resolution
488nm Laser/CH16, 638nm Laser/CH27* : ≧2.5% (HPCV)
Software
Unmixing algorithms Spectrum method (LSM, WLSM, PSA and Constraint option), Reverse matrix method (Conventional)
Autofluorescence Autofluorescence spectral detection
Virtual Filter Possible to change wavelength region for each fluorochromes in analysis
Export Data Format Flow Cytometry Standard (FCS) 3.0, 3.1
Main Unit
DimensionsMain unit: Width 60.0cm x Depth 63.5cm x Height 71.3cmFluidics cart: Width 78.6cm x Depth 52.1cm x Height 58.0cm
Weight Main unit: approx. 99kg (dried weight)/Fluidics cart: approx. 32kg (dried weight)
Air pressure supply 350kPa~450kPa (51psi~65psi)
AC Power supply AC100V 50/60Hz, AC120V 60Hz
Power consumption 220W (max)
Operating temperature 16-29 degrees Celsius, Room temperature variation: within 5 degrees Celsius
Operating humidity 20% to 80% (condensation free)
Recommended PC SP6800 workstation
Specifications
©2017 Sony Biotechnology Inc. All rights reserved. Sony, the Sony logo, and Flowpoint, are trademarks of Sony Corporation. eVolve is a trademark of and PE eFluor 610, eFluor 660, APC eFluor 780, and PerCP eFluor 710 are registered trademarks of eBioscience Corporation. Pacific Blue and Pacific Orange, are trademarks of and Alexa Fluor and Texas Red are registered trademarks of and licensed under patents assigned to Life Technologies Corporation. Qdot is a registered trademark of Invitrogen Corporation. Krome Orange, is a trademark of Beckman Coulter. BD Horizon, BD Horizon Brilliant and BB515 are trademarks of Becton Dickinson and Company. Brilliant Violet, Brilliant Violet 421, BV421, Brilliant Violet 570, BV570, Brilliant Violet 605, BV605, Brilliant Violet 650, and BV650, Brilliant Violet 510, BV510, Brilliant Violet 711, BV711, Brilliant Violet 785 and BV785 are trademarks of Sirigen Group Ltd. Vio is a registered trademark of Miltenyi Biotec GmbH. PE Dazzle 594 is a trademark of Biolegend. Cy and CyDye are registered trademarks of GE Healthcare. De Novo and FCS Express are trademarks of De Novo Software. All other trademarks are property of their respective owners. For non-clinical research use only. Not for use in diagnostic or therapeutic procedures, or for any other clinical purpose. The SP6800 Spectral Analyzer is a Class 1 laser product.
1.01.032916.4
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