GUAVA EASYCYTE SYSTEMS - dev.luminexcorp.com...Guava ® easyCyte Systems E tometry UNLEASH WHAT’S...
Transcript of GUAVA EASYCYTE SYSTEMS - dev.luminexcorp.com...Guava ® easyCyte Systems E tometry UNLEASH WHAT’S...
GUAVA® EASYCYTE SYSTEMS Expanding the potential of flow cytometry
The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
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Guava® easyCyte Systems │ Expanding the potential of flow cytometry
UNLEASH WHAT’S POSSIBLEOver fifteen years ago, Guava Technologies introduced the first compact benchtop flow cytometers. Today, the Guava® easyCyte line has been updated to offer up to 3 lasers and 14 parameters with greater sensitivity and optional high throughput capabilities. Powered by intuitive software, the Guava® easyCyte flow cytometers are some of the most dynamic and flexible benchtop systems available.
• Up to 3 lasers and 14 parameters on a benchtop instrument
• Microcapillary fluidics design eliminates sheath fluid and waste carboys
• Accurate absolute cell counts via microcapillary and positive displacement syringe pump
• Intuitive software includes comprehensive cell-health related assays
• Detection of particles as small as 0.2 μm
• High-throughput option for walk-away acquisition of up to 96 samples
Sheath Fluid: None
Waste
Laser
Flow
Cel
l
Sample
Sam
ple
Flow
Waste: < 50 mL/8 hour run
Typical # Cells:10,000 - 1,000,000 cells/mL
Microcapillary Flow Cytometry
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Inside the Guava® easyCyte 12HT systems
405
nmNIR
785/
70RE
D69
5/50
RED
-R66
1/15
YLW
583/
26G
RN52
5/30
488
nm
642
nmSSC
FSC
BLU448/50
How it Works
The Guava® easyCyte systems use patented, microcapillary, laser-based technology capable of detecting mammalian and microbial cells, particles, and beads. A sample of fluorescently labeled cells is aspirated into a uniquely proportioned microcapillary flow cell. Forward and side scatter characteristics are detected by photodiode, and fluorophores excited by the violet, blue, or red laser emit signals that are spectrally filtered to resolve up to 10 fluorophores simultaneously.
405 nm - Violet Laser
488 nm - Blue Laser
642 nm - Red Laser
Blu - V GRN - V YEL - V RED - V
GRN - B YEL - B RED - B NIR - B
RED - R NIR - R
(448/50)BV421™
Cascade Blue
(525/30)BV520™
Cascade Green
(583/26)BV605™
(695/50)BV650™
(525/30)FITC
AF® 488(583/26)
PE(695/50)PE-Cy5.5
(785/70)PE-Cy7
(661/15)APCCy5
(785/70)APC-Cy7
APC-AF® 750
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Guava® easyCyte Systems │ Expanding the potential of flow cytometry
Sensitive and specificSpherotech 8-color beads analyzed on the Guava® easyCyte 12HT system demonstrate the instrument’s proficiency for resolving adjacent fluorophores in multiple detection channels.
100
140 Cascade Blue MESF: 40Plot P02, gated on P01.R1
Blue - V Fluores (BLU-V-ALog)
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70
40
10
101 102 103 104 105
Count
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140PE-Cy5 MESF: 120Plot P05, gated on P01R1
Red - B Fluores (Red-B-ALog)
100
70
40
10
101 102 103 104 105
Cou
ntBlue Fluorescence - 405nm laser Red Fluorescence - 488nm laser
100
140PE-Cy7 MESF: 120Plot P06, gated on P01R1
Near IR-B Fluo...a (NIR-B-ALog)
100
70
40
10
101 102 103 104 105
Cou
nt
100
140APC MESF: 50Plot P07, ungated
RED-R Fluores... (RED-R-ALog)
100
70
40
10
101 102 103 104 105
Cou
nt
NIR Fluorescence - 488nm laser Red Fluorescence - 642nm laser
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140 FITC MESF: 25Plot P04, gated on P01. R1
Yellow-B Fluor...a (YEL-B-ALog)
100
Cou
nt
70
40
10
101 102 103 104 105 100
140 FITC MESF: 75Plot P03, gated on P01. R1
Green-B Fluor... (GRN-B-ALog)
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70
40
10
101 102 103 104 105
Cou
nt
Yellow Fluorescence - 488nm laser Green Fluorescence - 488nm laser
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Plot P04, gated on PO1.CD45+.CD3-.R9
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101
10
2
1
03
104
CD
19 B
V 5
10 (
GR
N-V
-HLo
g)
CD19+
CD16&CD56+
101 102 103
CD16 + CD56 PE (YEL-B-HLog)
10 μL adult human blood was stained for 20 minutes at room temperature with a cocktail containing anti-CD45 PerCP-Cy5.5, anti-CD3 Brilliant™ Violet 421, anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CD16+CD56 PE, anti-CD19 Brilliant™ Violet 510, anti-CD45 RA APC, and anti-CD62L FITC. After incubation, cells were lysed and fixed with 180 μL Guava® lysing solution for 15 minutes at room temperature. Samples were then acquired on the Guava® easyCyte 12HT system. Lymphocytes identified as CD45+ were selected and subsequently gated into a SSC vs. CD3 plot. T cells (CD3+ and CD45+) were gated into a CD4 vs CD8 plot. CD4+ and CD8 + T cells were subtyped by evaluating each population using CD45RA and CD62L to differentiate naive from memory cells. To distinguish natural killer (NK) and B cells, CD3-negative cells were gated into a plot comparing CD19 (B cells) and CD16+56 (NK cells).
Immunological Phenotyping
Plot P01, ungated
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0 1
0000
3
0000
5000
0
700
00
90
000
CD45 PerCP-Cy5.5 (RED-HLog)
CD45+
Sid
e Sca
tter
(SSC
-HLi
n)
Plot P02, gated on P01.CD45+
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0 1
0000
3
0000
5000
0
700
00
90
000
CD3 BV 421 (BLU-V-HLog)
Sid
e Sca
tter
(SSC
-HLi
n)
CD3- CD3+Plot P03, gated on PO1.CD45+.CD3+
101 102 103 104
CD8 APC-Cy7 (NIR-R-HLog)
101
10
2
103
10
4
CD
4 PE-C
y7 (
NIR
-B-H
Log)
CD4+
CD8+
Plot P05, gated on P01.CD45+.CD3+.CD
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CD45RA APC (RED-R-HLog)
CD
62L
FITC
(G
RN
-B-H
Log)
100
10
1
102
1
03
104
R6
Plot P06, gated on P01.CD45+.CD3+.CD
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CD45RA APC (RED-R-HLog)
CD
62L
FITC
(G
RN
-B-H
Log)
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101
10
2
1
03
104
R6
CD45+
CD3+
CD3-
CD4+
CD8+
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Guava® easyCyte Systems │ Expanding the potential of flow cytometry
1. Create and apply analysis methods across multiple dat asets.
2. Perform compensation during acquisition or analysis or use the automated compensation features.
3. Drag-and-drop gating.
4. View up to 24 plots at once.
5. Refine gates in real time.
6. Default or custom statistics.
7. Multiple plot and gating options.
8. Minimal gain adjustment needed when performing routine assays.
9. Analyze both tubes and plates.
SoftwareThe guavaSoft® operating system software provides access to modules for acquisition and analysis, as well as instrument setup and maintenance. The guavaSoft® system includes templates for use with a wide range of MilliporeSigma flow cytometry kits to simplify your experiments and data collection. Additionally, the guavaSoft® package includes InCyte™, an intuitive open software package for custom analysis. Results can be exported to spreadsheets or as industry-standard FCS 2.0 or 3.0 files for further analysis. GuavaSoft® software includes 21 CFR Part 11-enabling features.
InCyte™ Software: IntuitiveOur InCyte™ software has an intuitive, easy-to-use interface that enables you to focus on data at the sample or experimental level. The software simplifies setup and analysis of plots with drag-and-drop features, while automated compensation makes it easy to perform complex, multi-color assays. The instant update feature responds in real time to change analysis conditions for viewing. The multiparameter heat mapping function allows analysis of entire plates of data in the time previously required to analyze a single sample. These features provide a simple and rapid means to attain a macroscopic view of experiment “hits” and easily compare different experiments in real time. InCyte™ software is especially useful for interpreting the results of high-throughput cell-based assays.
Annexin V CF400 (GRN - HLog
7AAD
(RED
- H
Log)
Plot PO3, gated on P01.R1
Mito
Sens
e Re
d (R
ED2
- H
Log)
Annexin V CF488 (GRN - HLog
Plot PO2, gated on P01.R1Plot PO1, ungated
Forward Scatter (FSC - HLog)
Side
Sca
tter
(SSC
- H
Lin)
1 2 3 4 5 6 7 8 9 10 11 12
Count: 2746
100 101 102 103 104020
0040
0060
0080
00
A
B
C
D
E
F
G
H
MitoSense Red (+)Annexin V (-)68.46%
MitoSense Red (+)Annexin V (+)
4.44%
MitoSense Red (-)Annexin V (-)6.19%
MitoSense Red (-)Annexin V (+)
20.90%100 101 102 103 104
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10
1
102
10
3
104
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10
1
102
10
3
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1
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0 1
0 2
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0 4
0 5
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0 9
0
11
0
IC50=4.9µM
HeLa 24 hours
IC50 determination using the Cytochrome c Kit was analyzed with the built-in IC50/EC50 curve fitting feature of InCyte™ software. Cells were acquired on the Guava® easyCyte 8HT system. Plot A shows the drag-and-drop gating strategy used for the IC50
A.
Res
pon
se L
evel
, %
Res
pon
se L
evel
, %
0 1
0 2
0 3
0 4
0 5
0 6
0 7
0 8
0 9
0
11
0
10-1 100 101 102 103 10-1 100 101 102 103
Concentration Concentration
IC50=38µM
B. C.
HeLa cells in microtiter plates were treated with various cytotoxic compounds for 24 hours. Cells were stained using MilliporeSigma’s MitoDamage, MitoCaspase, or MitoStress kits. Cells were acquired on the Guava® easyCyte system and percent population data were compared in a heat map format using our InCyte™
InCyte™ Software Heat Map View
IC50 Determination within InCyte™ Softwaredetermination. Plot B shows the IC50 curve results for gambogic acid and Plot C shows the IC50 for etoposide. The once-complex task of generating the IC50 or EC50 curve for a given compound is automated by InCyte™ based on quantitation of fluorescent signal.
Software. The InCyte™ heat map function facilitated the rapid identification of compounds inducing positive results, by comparison of all 5 parameters simultaneously as shown in the pie charts above. The data show the results for cells treated with 80 different compounds in a single plate.
Combine groups of data to construct heat maps, IC50, or EC50 curves.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
0 -20%
20 -40%
40 -60%
60 -80%
80 -100%
MitoSense Red
MitoStress
Annexin V-CF488
Caspase 3/7
7-AAD
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Guava® easyCyte Systems │ Expanding the potential of flow cytometry
Small Particle DetectionThe Guava® easyCyte 8HT and 12HT systems have been shown to detect particles as small as 0.2 µm, a significant improvement over typical flow cytometers. This increased resolution and sensitivity means better separation, making gating and identification of dim populations easier. These capabilities may prove particularly useful for researchers analyzing particulates, beads, bacteria or algae.
Acquisition of a mixture of beads of known size demonstrates the ability of Guava® easyCyte 12HT instruments to detect and discriminate particles as small as 0.2 µm.
Lipid measurement of chlorophyll A-positive algae. Identification of algal cells containing chlorophyll A; chlorophyll A fluoresces in the red channel (A). Gate applied to select for chlorophyll A-positive cells (B). Histograms showing a wide range of lipid content (as evidenced by BODIPY green fluorescence intensity) for a variety of algal strains (C), with one clone showing as much as 500 times the lipid content as others.
Turning algae into biofuelsGuava® easyCyte systems are currently participating in algal biomass laboratories worldwide, where flow cytometry facilitates selection of high lipid content strains and efficient monitoring of cultures. Because microcapillary systems require smaller sample volumes, generate significantly less waste, have lower operating costs, enable high sample throughput, and have a small instrument footprint, they are a natural choice for demanding laboratory settings.
A. Not Gated B. Gated by Chlorophyll A
C. Histograms showing varying lipid content of different algae clones
Sid
e-S
catt
er (
SS
C-H
Log)
Forward Scatter (FSC-HLog)
1.3µm
0.8µm
0.4µm0.2µm
100
100 101 102 103 104 105
101
102
103
104
105
100 101 102 103 104
100
101
102
102
104
Chlorophyll A (RED-HLog)
Sid
e Sca
tter
(SSC
-HLo
g)
100 101 102 103 104
0
38
75
113
150
BODIPY (GRN-HLog)
Cou
nt
M1 M2
100 101 102 103 104
0
5
10
20
30
BODIPY Fluorescence
Cou
nt
High LipidContent Clone
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Mouse peripheral blood cells gated on lymphocytes (left) were stained with Milli-Mark® FCMAB244CP5 anti-mouse CD4 (clone GK1.5) PerCP/Cy5.5. CD4+Th1 cells were identified (right) by costaining with FCMAB243P, anti-mouse IFN gamma (clone XMG1.2) PE. Data were analyzed with InCyte™ software on a Guava® easyCyte HT cytometer.
Uninduced
MitoS
ense
Red
MitoS
ense
Red
Annexin V, CF488A
7-AAD
Red
2 Fl
uore
sece
nce
(R
D2-
HLo
g)Red
2 Fl
uore
sece
nce
(R
D2-
HLo
g)
Green Fluorescence (GRN-HLog)
Red Fluorescence (RED-HLog)
Green Fluorescence (GRN-HLog)
Red Fluorescence (GRN-HLog)
94.4%
0.75% 3.7%
1.1%
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100
100
100
101
101
101
101
102
102
102
102
103
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103
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100
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100
100
101
101
101
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102
102
102
103
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103
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104
104
104
54.7%
14.6% 27.0%
3.7%
2 µM Staurosporine
95.2%
3.2% 1.3%
0.3%
Depolarized Cells
Dead Cells
Live Cells58.1% 0.08%
41.0% 0.8%
Plot P02, gated on P01.Live
101 102 103 104
CD4-PerCP (RED-HLog)
IFN
g-P
E (
YEL-
HLo
g)
100
101
102
103
104
Plot P03, gated on P04.R1.R2
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0 10
000
30
000
500
00 7
0000
9
0000
CD4 (RED-HLog)
Sid
e Sca
tter
(SSC
-HLi
n)
FlowCellect® Flow Cytometry Kits MilliporeSigma’s optimized, turnkey assay kits reduce sample preparation time, minimize assay development and simplify data analysis.
We offer more than 40 FlowCellect® kits optimized for key assays in cell health, immunology and cell signaling.
The FlowCellect® MitoDamage Kit for flow cytometry contains MitoSense Red, a fluorescent cationic dye that accumulates in the mitochondria and is responsive to changes in mitochondrial potential, a hallmark of early apoptosis.
Flow Cytometry Reagents
Fluorescence-conjugated Flow Cytometry AntibodiesChoose from our growing portfolio of over 1500 conjugated antibodies, including antibodies specific to many CD markers and immune signaling targets. With the reliability and quality of Upstate, Chemicon and Calbiochem, MilliporeSigma’s conjugated antibodies validated for flow cytometry are available in multiple colors, to enable you to unequivocally discriminate cell subpopulations:
All of our flow-validated antibodies come with our 100% performance and satisfaction guarantee.
Our diverse portfolio of reagents and assays facilitate fluorescence-based detection of proteins and nucleic acids, and have been validated for use on the Guava® easyCyte instrument platform.
Dot plots depicting Jurkat cells treated with multiple inducers and stained using MitoDamage kit. Dot plots of cells treated or not with staurosporine to induce apoptosis and acquired on the Guava® easyCyte 12HT system show percentages of cells positive for MitoSense Red (left). The guavaSoft® MitoDamage module facilitates experiment-level assessment of changes in mitochondrial potential that signal early apoptosis via heat mapping of up to 96 samples.
• FITC
• APC
• PE-Cy7
• PerCP-Cy5.5
• PE
• PE-Cy5
• APC-Cy7
• Violet450
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Guava® easyCyte Systems │ Expanding the potential of flow cytometry
Guava® easyCyte Single Sample System 5 5 HPL 6-2L 8
Catalog No. 0500-5005 0500-5009 0500-5007 0500-5008
Violet (405 nm) Laser
Blue (488 nm) 50 mW Laser
Blue (488 nm) 150 mW Laser
Green (532 nm) Laser
Red (642 nm) Laser
FSC
SSC
Blue-V (450/45 nm)
Green-V (512/18 nm)
Green-V (525/30 nm)
Yellow-V (575/25 nm)
Yellow-V (583/26 nm)
Orange-V (620/52 nm)
Red-V (695/50 nm)
NIR-V (785/70 nm)
Green-B (512/18 nm)
Green-B (525/30 nm)
Yellow-B (575/25 nm)
Yellow-B (583/26 nm)
Red-B (695/50 nm)
NIR-B (785/70 nm)
Yellow-G (575/25 nm)
Orange-G (609/30 nm)
Orange-G (620/52 nm)
Red-G (695/50 nm)
NIR-G (785/70 nm)
Red-R (664/20 nm)
NIR-R (785/70 nm)
Microcapillary Fluidics
Direct, Absolute Cell Counts
Automation-plate and tubes
Mixing
Dell® Laptop
InCyte™ Software
Digital Signal Processing
Guava® easyCyte Single Sample Systems
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Guava® easyCyte HT Systems
Guava® easyCyte HT System 5HT 5HT HPL 6HT-2L 8HT 11HT 12HT BG HT BGR HT BGV HT
Catalog No. 0500-4005 0500- 4009 0500-4007 0500-4008 0500- 4020 0500-4012 0500-4015
0500-4025 0500-4030
Violet (405 nm) Laser
Blue (488 nm) 50 mW Laser
Blue (488 nm) 150 mW Laser
Green (532 nm) Laser
Red (642 nm) Laser
FSC
SSC
Blue-V (450/45 nm)
Green-V (512/18 nm)
Green-V (525/30 nm)
Yellow-V (575/25 nm)
Yellow-V (583/26 nm)
Orange-V (620/52 nm)
Red-V (695/50 nm)
NIR-V (785/70 nm)
Green-B (512/18 nm)
Green-B (525/30 nm)
Yellow-B (575/25 nm)
Yellow-B (583/26 nm)
Red-B (695/50 nm)
NIR-B (785/70 nm)
Yellow-G (575/25 nm)
Orange-G (609/30 nm)
Orange-G (620/52 nm)
Red-G (695/50 nm)
NIR-G (785/70 nm)
Red-R (664/20 nm)
NIR-R (785/70 nm)
Microcapillary Fluidics
Direct, Absolute Cell Counts
Automation-plate and tubes
Mixing
Dell® Laptop
InCyte™ Software
Digital Signal Processing
Description Cat. No.
Single Sampling Instruments
Guava® easyCyte 5 Base System 0500-5005
Guava® easyCyte 5HPL Base System 0500- 5009
Guava® easyCyte 6-2L Base System 0500-5007
Guava® easyCyte 8 Base System 0500-5008
High Throughput Sampling Instruments
Guava® easyCyte 5HT Base System 0500-4005
Guava® easyCyte 5HT HPL Base System 0500- 4009
Guava® easyCyte 6HT-2L Base System 0500-4007
Guava® easyCyte 8HT Base System 0500-4008
Guava® easyCyte HT BG Base System 0500-4015
Guava® easyCyte 11HT Base System 0500-4020
Guava® easyCyte HT BGR Base System 0500-4025
Guava® easyCyte 12HT Base System 0500-4012
Guava® easyCyte HT BGV Base System 0500-4030
Ordering Information
Ver. 1.02018 - 09556
04/2018
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M, FlowCellect, Guava, InCyte and guavaSoft are registered trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.
MilliporeSigma400 Summit DriveBurlington, MA 01803
To place an order or receive technical assistanceIn the U.S. and Canada, call toll-free 1-800-645-5476 For other countries across Europe and the world, please visit: EMDMillipore.com/offices For Technical Service, please visit: EMDMillipore.com/techservice
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