SP6800 Spectral Analyzer

Transcription

1 SP68 Spectral Analyzer Sony Biotechnology Inc.

2 SP68 Spectral Analyzer The Sony SP68 Spectral Analyzer uses spectral technology to optimize sensitivity and enhance dim signal detection by collecting photons from 42nm to 8nm. Spectral technology also simplifies multicolor panel design, by eliminating bandpass 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 Flowpoint TM 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 Express TM 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 SP68 System Overview The 45nm, 488nm and 638nm excitation lasers are positioned to reduce fluorescent noise. They enable the system to support 16 or more fluorescent parameters. The SP68 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. 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 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 analysis Forward and Side Scatter parameters to allow relative size and complexity measurements. x1, 4 SSC_A FSC_A 6 x1, Emitted light is directed through through a 32 Channel PMT that produces 66 data points of signal detection to analyze emitted photons from 42nm to 8 nm to ensure accurate visualization. A unique prism collection system Delivers light through 1 consecutive prisms allowing optimal signal separation while minimizing light loss. 3

4 Software The SP68 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. System Start Up 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. Standardization 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. When engaged, standardization mode sets the SP68 to predetermined global settings that eliminate instrument variability. 4

5 Spectral Library The Spectral Library lets users create a personal reagent library that simplifies experiment 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 available to users with a simple click improving accuracy and streamline panel design. Experiment Creation 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 experiment 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 singlestained or multicolor assay simplifying experiment creation. Acquisition Functions and Analysis All acquisition functions, including instrument 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 (25ul/min) offering flexibility. System Shutdown On shutdown, the SP68 software guides the user though pre shutdown cleaning and shuts down the instrument automatically. Software wizards are also available to guide users through Bleach Cleaning and Rinse procedures. 5

6 FCS Express from De Novo Software Spectral Analyzers from Sony include FCS Express, from De Novo Software. FCS Express offers a range of new analysis tools from live gating to batch analysis. Native support for Sony Spectral data files to enable sophisticated data transformations and visualizations such as spectral overlays, tsne, Spade, and heat maps. Live Gating 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 Intensity 1 7 FOXP3 + FOXP Height (Wavelength) FOXP3 : PE-Dazzle594 - Area - CD4 : BV421 - Area SSC - Area (x 1) Lymphs CD45 : BV711 - Area Intensity 1 7 FOXP3 + FOXP Height (Wavelength) FOXP3 + FOXP Height (Wavelength) Intensity FOXP3 : PE-Dazzle594 - Area - - CD4 : BV421 - Area Flexible Analysis Batch and Report 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. 6

7 Spectral Analysis Technology Spectral analysis technology is the foundation of the SP68 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. LD-488_A E 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 fingerprint of each fluorochrome from 42nm to 8nm Wavelengths Visualization of fluorescent cell populations using the analyzer s unique prism collection system. Spectral Unmixing 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 for complete and optimal visualization of fluorochromes. 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 42nm to 8nm. In doing so it enhances dim signal detection for better visualization of rare populations, fluorescent proteins and fluorochromes excited by multiple lasers. CD4_APC_A E: 95.81% G: 2.18% F: 1.28% Unmixing CD4_APC_A E: 95.65% G: 8.63% F: 1.42% 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. - 1 IL2_PE_A A Unstained cells High Auto-fluorescence B - 1 PE Positive cells IL2_PE_A 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 SP68. LD-488_A E Wavelengths LD-488_A G Wavelengths LD-488_A F Wavelengths 7

8 Uniform measurement of Fluorescent Emissions 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 42nm to 8nm. 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. Pre correction Post correction Pre-correction Post-correction LD488_H Correction LD488_H Channel Channel PE-Cy5 PE-Cy5 Pre and Post Correction Profile. Each graph illustrates pre and post correction in the SP68 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. Subtracting Auto-Fluorescence Improves Visualization 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 autofluorescence include cell fixation and permeabilization. Using spectral technology, auto-fluorescent spectral fingerprints can be subtracted to allow researchers to see the true fluorescent population. Alexa Fluor 7_A A. With Auto-fluorescence H-Q1: 88.2% H-Q2: 3.5% Alexa Fluor 7_A B. Without Auto-fluorescence H-Q1: 91.66% H-Q2:.4% Activated T Cells with GFP and Alexa Fluor H-Q3: 8.33% H-Q4:.15% H-Q3: 8.24% H-Q4:.7% - GFP_A - GFP_A C. With Auto-fluorescence D. Without Auto-fluorescence B22_BV65_A -1 1 O T:.39% Y:.27% S:.3% LD45/638_A S LD45/638_A V - LD45/638_A Y - SSC_A GFP Pos:.9% SSC_A GFP Pos:.21% Liposomes with GFP - R: 95.62% F4/8_PE_A Wavelength Wavelength Wavelength 1 1 GFP neg: 98.74% 1 1 GFP neg: 99.66% - GFP_A - GFP_A Unstained mouse splenocytes were analyzed with the SP68 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 7 were analyzed with the SP68. A double positive population is present in the uncorrected density plot. B. This double positive population disappears when the autofluorescent 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. 8

9 Fluorochrome Panel Guide Application data dye options and combinations. Select one from each group. Laser 488nm 5 wavelength (nm) Group 1 Group 2 Group 3 Group 4 Alexa Fluor 488; 488nm Alexa Fluor 532; 488nm PE; 488nm PE DazzleTM 594; 488nm BrilliantTM Blue 515; 488nm Alexa Fluor 514; 488nm PE Texas Red ; 488nm FITC; 488nm wavelength (nm) 5 6 Group 9 Group 1 Group 11 Group 12 Brilliant VioletTM 421; 45nm Pacific BlueTM ; 45nm Brilliant Violet 51TM ; 45nm Brilliant Violet 57TM ; 45nm Alexa Fluor 45; 45nm HorizonTM V45; 45nm AmCyan; 45nm Pacific OrangeTM ; 45nm Laser Horizon V5; 45nm 45nm Krome OrangeTM ; 45nm 638nm 9

10 Group 5 Group 6 Group 7 Group 8 PE Alexa Fluor 61; 488nm PE Cy5TM ; 488nm PerCP Cy5.5; 488nm PerCP efluor 71; 488nm PE Cy5.5; 488nm PerCP; 488nm PE Alexa Fluor 7; 488nm PE Cy7; 488nm PE Vio 77; 488nm Group 13 Group 14 Group 15 Group 16 Brilliant Violet 65TM ; 45nm Brilliant Violet 65TM ; 45nm Brilliant Violet 711TM ; 45nm Brilliant Violet 785TM ; 45nm Qdot 65; 45nm Qdot 655; 45nm Qdot 75; 45nm Qdot 8; 45nm evolvetm 65; 45nm efluor 66; 45nm Alexa Fluor 7; 638nm APC Cy7; 638nm Alexa Fluor 647; 638nm APC Cy5.5; 638nm APC Alexa 75; 638nm APC; 638nm APC efluor 78; 638nm Cy5; 638nm 1

11 Sample Data 16 color panel on Human Peripheral Blood Application data using 2 lasers 488nm Laser 45nm Laser CD3 Alexa Fluor 488 HLA-DR Pacific Blue CD8 PE CD38 BV421 CD4 PE Dazzle 594 CD24 PE Alexa Fluor 61 CD45RA CD33 BV51 BV57 CD19 CD2 PE Cy5 PE Cy CD16 CD11b BV65 BV CD13 PerCP efluor 71 CD11c BV711 CD45 PE Cy7 CD14 BV785 A B C D E x1, SSC_A Granulocytes Monocytes CD13_PerCP eflour71_a Granulocytes CD24-CD13 CD35_BV421_A - CD35-CD3 CD35-CD % CD16_BV65_A - CD35-CD3 Natural Killer Cells CD16_BV65_A - CD16-CD3: 1.73% F - - CD45_PE-Cy7_A CD45+ G CD24_PE-AF61_A CD11c-CD14- H - 1 CD3_Alexa Fluor 458_A CD19-CD2 I CD3_Alexa Fluor 458_A PLASMA B Cells - -1 CD3_Alexa Fluor 458_A CD19c-CD2 PLASMA B Cells CD14_BV785_A - - CD11c-CD14- CD23_PE-Cy5.5_A - CD35_BV421_A CD11b_BV65_A Memory B Cells Naive B Cells J - - CD11c_BV711_A Monocytes K CD4 - CD19_PE-Cy5_A L - HLA-DR_PacificBlue_A 1 CD45RA_BV51_A CD13-CD14 CD14 positive CD14_BV785_A CD14_BV785_A CD14_BV785_A - CD3-CD CD11c_BV711_A - CD13_PerCP eflour71_a - - CD11c_BV711_A Examples of the resolution of several important cell populations using only two lasers with the SP68. Clear population resolution is obtained with highly overlapping fluorochomes such as PE-Cy5/ PE-Cy5.5 (G) and Pacific Blue/BV421 (H). 11

12 16 color stained sample of Human Peripheral Blood Application data using 3 lasers 488nm Laser 45nm Laser CD3 Alexa Fluor 488 HLA-DR Pacific Blue CD8 PE CD5 BV421 CD4 PE Dazzle 594 CD24 PE Alexa Fluor 61 CD56 CD33 BV51 BV57 CD19 CD2 PE Cy5 PE Cy5.5 CD BV65 CD13 PerCP efluor nm Laser CD45 PE Cy7 CD38 APC CD11c Alexa Fluor 7 CD45RA APC-Cy Granulocytes CD45+ x1, SSC_A 4 2 Granulocytes Monocytes CD13_PerCP eflour71_a CD13+CD24+ Grans CD16_BV65_A - NK Cells CD2_PE-Cy5.5_A - - CD19+CD CD45_PE-Cy7_A CD24_PE-AF62_A CD3_Alexa Fluor 488_A - - CD19_PE-Cy5_A CD19+CD2+ Cytotoxic T Cells Monocytes CD38_APC_A Plasma B Cells: 99.57% CD8_PE_A - Cytotoxic T Cells CD45RA_APC-Cy7_A - Cytotoxic Naive T Cells Cytotoxic Effector T Cells CD16_BV65_A - CD16 positive HLA-DR_PacificBlue_A CD3_Alexa Fluor 488_A - CD38_APC_A - -1 CD3_Alexa Fluor 488_A Monocytes Granulocytes Helper T Cells: 5.38% Helper Naive T Cells CD13+CD33+ Monocytes CD4_PEDazzle 594_A CD45RA_APC-Cy7_A - Helper Effector T Cells CD24_PE-AF61_A - - CD5+CD24+ B Cells CD33_BV57_A CD38_APC_A - - CD24+CD38- Granulocytes CD3_Alexa Fluor 488_A - CD38_APC_A - - CD5_BV421_A - - CD13_PerCP eflour71_a CD24_PE-AF61_A 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 SP68 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. 12

13 Sample Data Application Data: Brilliant Violet Dyes Spectral Analysis allows multi-laser excited fluorochromes to be run without using a complicated compensation matrix. BV421 BV51 BV57 BV65 BV65 BV711 BV A B C D E H CD45+ CD45+ CD45+ CD45+ x1, 1 CD45+ 8 SSC_A CD8_BV57_A CD4_BV51_A CD16/56_BV65_A - CD3_BV421_A CD45_BV711_H - CD3_BV421_A - CD3_BV421_A CD19_BV65_A - -1 CD14_BV785_A 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 BV65 and BV65 (D). 12-color Staining of Human Peripheral Blood Leukocytes Example of Human Peripheral Blood Leukocytes was analyzed on the SP68 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. Marker Fluorochrome 488nm Laser 45/638nm Laser CD45RA Alexa Fluor 488 TCR-gd CD56 CD45 PE PE-Dazzle594 PerCP-Cy5.5 LD488_H LD45/638_H CD3 PE-Cy7 CCR7 CD27 CD33 BV421 BV51 BV Wavelength Wavelength CD4 BV65 CD19 APC CD8 Alexa Fluor 7 HLADR APC-Cy7 13

14 CD27-BV51 13 CD19-APC 13 TCR GD-PE 13 CD56-PE Dazzle CD45RA-AF CCR7-BV CD56-PE Dazzle HLA-DR-APCCy CD45RA: Alexa Fluor Area 14 CCR7: BV421 - Area CD56: PE-Dazzle594 - Area HLADR: APC-Cy7 - Area SSC - Area (x 1) SSC-A (x 1) Sony SP68 Spectral Analyzer CD45: PerCP-Cy5.5 - Area Conventional Flow Cytometer CD4: BV65 - Area CD4-BV CD8: Alexa Fluor 7 - Area Conventional Flow Cytometer CD8-AF CD56-PE Dazzle CD56: PE-Dazzle594 - Area CD19: APC - Area CD19-APC Sony SP68 Spectral Analyzer Conventional Flow Cytometer HLADR: APC-Cy7 - Area HLA-DR-APCCy TCR-gd: PE - Area TCR GD-PE CD27: BV51 - Area CD27-BV CD45-PerCPCy5.5 CD3-PECy CD3-PECy7 14 CD3-PECy CD3-PECy7 CD3-PECy CD3-PECy7 14 CD3-PECy7 3 Sony SP68 Spectral Analyzer 4 Sony SP68 Spectral Analyzer CD27: BV51 - Area CD4: BV65 - Area TCR-gd: PE - Area CD56: PE-Dazzle594 - Area CD8: Alexa Fluor 7 - Area CD33: BV57 - Area CD33: BV57 - Area CD33: BV57 - Area CD33: BV57 - Area -13 Conventional Flow Cytometer Conventional Flow Cytometer Conventional Flow Cytometer CD27-BV CD4-BV TCR GD-PE CD56-PE Dazzle CD3-PECy7 CD3-PECy CD8-AF7 14 CD19-APC CD33-BV CD33-BV57 CD33-BV CD33-BV57 5 Sony SP68 Spectral Analyzer CD27: BV51 - Area CD19: APC - Area 15 TCR-gd: PE - Area CD56: PE-Dazzle594 - Area CD4: BV65 - Area CD4: BV65 - Area CD4: BV65 - Area CD4: BV65 - Area Conventional Flow Cytometer CD4-BV65 CD4-BV65 CD4-BV65 CD4-BV

15 Sample Data Application Data: Dyes with issues 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 SP68 is capable of analyzing all of these by looking at all photons from 42nm to 8nm and unmixing each unique spectral fingerprint. CD8_BV421_A Cells CD4_PacificBlue_A Example 1: Pacific Blue (457nm emission) and Brilliant Violet 421 (422nm emission) APC excitation APC emission PE Cy5 excitation PE Cy5 emission CD8_PE-Cy5_A E CD4_APC_A Example 2: PE Cy5 (excited with 488nm and 638nm lasers) and APC (excited with 638nm laser) A LD488_H GFP_A Wavelength Example 3: Fluorescent GFP and YFP need no special bandpass filter set with the SP68 Normal PE Cy7 C LD488_A PE Cy7 falling apart C Wavelength F LD488_A Unmixing PE_A Wavelength PE-Cy7_A Example 4: Spectral Analysis unmixes the spectral fingerprint the spectral fingerprint of PE Cy7 tandem to produce excellent resolutions. 15

16 Specifications Configuration 2 laser model, 488/638 2 laser model, 45/488 3 laser model,45/488/638 Model Number LE-SP68ZB LE-SP68ZC LE-SP68ZE Laser specification Lasers Semiconductor 2 laser model 488nm, 638nm Semiconductor 2 laser model 45/488 Semiconductor 3 laser model, 45/488/638 Maximum power (at flow cell) 488nm 4mW 638nm 6mW 45nm 6mW 488nm 4mW 45nm 6mW, 488nm 4mW 638nm 6mW Optics Irradiation form Detection optics Scatter signals Spectroscopic method 2-spot dual-axis irradiation Forward scatter (FSC), Side scatter (SSC) Prism array spectroscopy Fluorescent channels 32 channel PMT (wavelength: 5-8nm) 32 channel PMT (wavelength: 5-8nm) PMT x 2 (42-44nm, 45-47nm) Signal resolution Height 2 bit, Area 32 bit Sampling frequency: 5MHz System Measurement parameters Pulse shape parameters 64 channels, FSC, SSC, cell position XY Height, Area, Width 66 channels, FSC, SSC, cell position XY Fluidics Optical alignment Event Rate Sample loader type Supporting sample tube Sheath Flow Speed Detection channel dimensions Fluorescence sensitivity Automated alignment with Corefinder TM technology 1,eps (standard)/2,eps (maximum) Single auto-loader 5ml polystyrene round tubes Low (approx. 3m/s), Mid (approx. 5m/s), High (approx. 1m/s) 2μm x 2μm FITC: 12 MESF / PE: 7 MESF Performance Linearity FITC R / PE R Detection size range.5~4µm (beads) Software Fluorescence detection resolution Unmixing algorithms Autofluorescence Virtual Filter 488nm Laser/CH16, 638nm Laser/CH27* : 2.5% (HPCV) Spectrum method (LSM,, PSA and Constraint option), Reverse matrix method (Conventional) Autofluorescence spectral detection Possible to change wavelength region for each fluorochromes in analysis Export Data Format Flow Cytometry Standard (FCS) 3., 3.1 Dimensions Main unit: Width 6.cm x Depth 63.5cm x Height 71.3cm Fluidics cart: Width 78.6cm x Depth 52.1cm x Height 58.cm Main Unit Weight Air pressure supply AC Power supply Power consumption Operating temperature Operating humidity Recommended PC Main unit: approx. 99kg (dried weight)/fluidics cart: approx. 32kg (dried weight) 35kPa~45kPa (51psi~65psi) AC1V 5/6Hz, AC12V 6Hz 22W (max) degrees Celsius, Room temperature variation: within 5 degrees Celsius 2% to 8% (condensation free) SP68 workstation * Except for LE-SP68ZC (This HPCV value is calculated for red laser. )

17 North America/International Japan Europe 173 North First Street San Jose, CA U.S.A. Voice: FAX: , Konan, Minato-Ku, Tokyo, Japan Tel: Fax: The Heights, Brooklands. Weybridge, Surrey, KT13 XW, UK 217 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 61, efluor 66, APC efluor 78, and PerCP efluor 71 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 57, BV57, Brilliant Violet 65, BV65, Brilliant Violet 65, and BV65, Brilliant Violet 51, BV51, 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 SP68 Spectral Analyzer is a Class 1 laser product