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Guava EasyCyte SystemsExpanding the potential offlow cytometryGuava easyCyte Systems Flexible Intuitive Affordable

Unleash what’s possibleFifteen years ago, Guava Technologies introduced the first compactbenchtop flow cytometers. Today, the guava easyCyte line has beenupdated to offer up to 3 lasers and 12 parameters with greater sensitivityand optional high throughput capabilities. Powered by intuitive software,the guava easyCyte flow cytometers are some of the most dynamic andflexible benchtop systems available.Microcapillary Flow CytometryWasteSheath Fluid: None- Up to 3 lasers and 12 parameters on a benchtop instrument- Detection of particles as small as 0.2 μmWaste: 50 mL/8 hour run- Microcapillary fluidics design eliminates sheath fluid and waste carboysTypical # Cells:10,000 - 1,000,000 cells/mL- Intuitive software includes comprehensive cell-health related assaysLaserFlow CellSample Flow - High-throughput option for walk-away acquisition of up to 96 samplesSample

633 nm488 nm405 nmGR e the guava easyCyte 12 systemsHow it WorksThe guava easyCyte systems use patented, microcapillary,laser-based technology capable of detecting mammalian andmicrobial cells and beads. A sample of fluorescently labeledcells is aspirated into a uniquely proportioned microcapillaryflow cell. Forward and side scatter characteristics are detectedby photodiode, and fluorophores excited by the violet, blue, orred laser emit signals that are spectrally filtered to resolve upto 10 fluorophores simultaneously.SSCBLU448/50FSC405 nm - Violet Laser488 nm - Blue Laser633 nm - Red LaserBlu - VGRN - VYEL - VRED - V(448/50)BV421 Cascade Blue(525/30)BV520 Cascade Green(583/26)BV605 (695/50)BV650 GRN - BYEL - BRED - BNIR - B(525/30)FITCAF 488(583/26)PE(695/50)PE-Cy5.5(785/70)PE-Cy7RED - RNIR - R(661/15)APCCy5(785/70)APC-Cy7APC-AF 750

Sensitive and specificSpherotech 8-color beads analyzed on the guava easyCyte 12 system demonstrate the instrument’s proficiency forresolving adjacent fluorophores in multiple detection channels.Blue Fluorescence - 405nm laserGreen Fluorescence - 488nmCascade Blue MESF: 40Yellow Fluorescence - 488nmFITC MESF: 75FITC MESF: 10¹10²10³10⁴Blue - V Fluores 10⁵NIR Fluorescence - 488nm laserPE-Cy5 MESF: 120PE-Cy7 MESF: 0³10⁴Red - B Fluores (Red-B-ALog)10⁵10³10⁴10⁵APC MESF: 501404010²Red Fluorescence - 633nm laser1407010¹Yellow-B Fluor.a (YEL-B-ALog)Green-B Fluor. (GRN-B-ALog)Red Fluorescence - 488nm laser7070401010⁰10¹10²10³10⁴10⁵Near IR-B Fluo.a (NIR-B-ALog)1010⁰10¹10²10³10⁴RED-R Fluores. (RED-R-ALog)10⁵

Immunological Phenotyping1010103CD45 PerCP-Cy5.5 (RED-HLog)2101102103CD3 BV 421 (BLU-V-HLog)CD4 R6101102103CD45RA APC (RED-R-HLog)104CD3 104104CD3-Plot P04, gated on PO1.CD45 .CD3-.R9CD62L FITC (GRN-B-HLog)101102103CD3 100CD45 1CD3-Plot P03, gated on PO1.CD45-.CD3 104CD45 Plot P05, gated on P01.CD45 .CD3 .CD104Plot P02, gated on P01.CD45 Side Scatter (SSC-HLin)0 10000 30000 50000 70000 90000Side Scatter (SSC-HLin)0 10000 30000 50000 70000 90000Plot P01, ungatedPlot P06, gated on P01.CD45 .CD3 .CD101102103CD16 CD56 PE (YEL-B-HLog)10 μL adult human blood was stained for 20 minutes at room temperaturewith 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 lysingsolution for 15 minutes at room temperature. Samples were then acquired onthe guava easyCyte 12HT system. Lymphocytes identified as CD45 wereCD62L FITC (GRN-B-HLog)101102103CD4 CD8 101102103CD8 APC-Cy7 (NIR-R-HLog)104100CD16&CD56 CD4 PE-Cy7 (NIR-B-HLog)102103CD19 101100CD19 BV 510 (GRN-V-HLog)101102103CD8 R6101102103CD45RA APC (RED-R-HLog)selected and subsequently gated into a SSC vs. CD3 plot. T cells (CD3 andCD45 ) were gated into a CD4 vs CD8 plot. CD4 and CD8 T cellswere subtyped by evaluating each population using CD45RA and CD62L todifferentiate naive from memory cells. To distinguish natural killer (NK) andB cells, CD3-negative cells were gated into a plot comparing CD19 (B cells)and CD16 56 (NK cells).104

SoftwareInCyte Software: IntuitiveThe guavaSoft operating system software provides access to modulesfor acquisition and analysis, as well as instrument setup and maintenance.The guavaSoft system includes templates for use with a wide range ofEMD Millipore flow cytometry kits to simplify your experiments and datacollection. Additionally, the guavaSoft package includes InCyte software, an intuitive open software package for custom analysis. Resultscan be exported to spreadsheets or as industry-standard FCS 2.0 or3.0 files for further analysis. GuavaSoft software includes 21 CFR Part11-enabling features.EMD Millipore’s InCyte software has an intuitive, easy-to-use interfacethat enables you to focus on data at the sample or experimental level.The software simplifies setup and analysis of plots with drag-and-dropfeatures, while automated compensation makes it easy to perform complex,multi-color assays. The instant update feature responds in real time tochange analysis conditions for viewing. The multiparameter heat mappingfunction allows analysis of entire plates of data in the time previouslyrequired to analyze a single sample. These features provide a simple andrapid means to attain a macroscopic view of experiment “hits” and easilycompare different experiments in real time. InCyte software is especiallyuseful for interpreting the results of high-throughput cell-based assays.Perform compensation duringacquisition or analysis or usethe automated compensationfeatures.View up to 24 plotsat once.Drag-and-drop gating.Refine gates in real time.Plot PO2, gated on P01.R1MitoSense Red ( )Annexin V ( )4.44%Default orcustomstatistics.10310140001026000Side Scatter (SSC - HLin)20001101102103Forward Scatter (FSC - HLog)234567100100MitoSense Red (-)Annexin V (-)6.19%1001011048910A1112102MitoSense Red (-)Annexin V ( )20.90%103104Annexin V CF488 (GRN - HLog1040Plot PO3, gated on P01.R1Multiple plotand gatingoptions.7AAD (RED - HLog)103BC102DEMinimal gainadjustment neededwhen performingroutine assays.101FG100Create andapply analysismethods acrossmultiple datasets.MitoSense Red ( )Annexin V (-)68.46%MitoSense Red (RED2 - HLog)8000104Count: 2746Plot PO1, ungatedH100Analyze both tubesand plates.101102103Annexin V CF400 (GRN - HLog104

InCyte Software Heat Map ViewHeLa 24 hours123456789101112ACombine groups of datato construct heat maps,IC50, or EC50 pase3/7F7-AADG020%HHeLa cells in microtiter plates were treated with various cytotoxic compoundsfor 24 hours. Cells were stained using EMD Millipore’s MitoDamage,MitoCaspase, or MitoStress kits. Cells were acquired on the guava easyCyte system and percent population data were compared in a heat map formatusing EMD Millipore’s InCyte Software. The InCyte heat map function20 40%40 60%60 80%80 100%facilitated the rapid identification of compounds inducing positiveresults, by comparison of all 5 parameters simultaneously as shown inthe pie charts above. The data show the results for cells treated with80 different compounds in a single plate.IC50 Determination within InCyte Softwaregambogic acid and Plot C shows the IC50 for etoposide. The once-complextask of generating the IC50 or EC50 curve for a given compound is automatedby InCyte based on quantitation of fluorescent signal.IC50 determination using the Cytochrome c Kit and analyzed with the built-inIC50/EC50 curve fitting feature of InCyte software. Cells were acquired on theguava easyCyte 8HT system. Plot A shows the drag and drop gating strategyused for the IC50 determination. Plot B shows the IC50 curve results forB.100Response Level, %Response Level, %10-1IC50 38µM0 10 20 30 40 50 60 70 80 90 110C.0 10 20 30 40 50 60 70 80 90 110A.101Concentration10210310-1IC50 4.9µM100101Concentration102103

Small Particle DetectionTurning algae into biofuelsThe guava easyCyte 8 and 12 systems have been shown to detect particlesas small as 0.2 µm, a significant improvement over typical flow cytometers.This increased resolution and sensitivity means better separation, makinggating and identification of dim populations easier. These capabilities mayprove particularly useful for researchers analyzing particulates, beads,bacteria or algae.easyCyte systems are currently participating in algal biomass laboratories10worldwide, where flow cytometry 10facilitatesselection of high lipid contentstrains and efficient monitoring of cultures. Because microcapillary systems1010require smaller sample volumes, generatesignificantly less waste, have lower10operating costs, enable high sample10 throughput, and have a small instrumentfootprint, they are a natural choice for demanding laboratory settings.21Side Scatter (SSC-HLog)Side Scatter (SSC-HLog)2221100100010101100A. Not GatedPlot 4: No Gated0.8µm10 ll A (RED-HLog)M138010010100104 310104100101M2M11011023102 10BODIPY (GRN-HLog)BODIPY (GRN-HLog)M24103 10104Plot 4: Gated by Chlorophyll AC.150 Histograms showing10 010110 210 310 410 5Forward Scatter (FSC-HLog)30107530201055M2M13801000101102BODIPY (GRN-HLog)1030100104101102103101 Fluorescence102BODIPYBODIPY Fluorescence103104Lipid measurement of chlorophyll A-positive algae. Identification of algal cellscontaining chlorophyll A; chlorophyll A fluoresces in the red channel (A). Gate30applied to select for chlorophyll A-positive cells (B). Histograms showingHigh Lipid20Content Clone by BODIPY green fluorescencea widerange of lipid content (as evidenced10intensity)for a variety of algal strains (C), with one clone showing as muchas 500times the lipid content as others.5CountAcquisition of a mixture of beads of known size demonstrates the ability ofguava easyCyte 12 instruments to detect and discriminate particles assmall as 0.2 µm.100High LipidHigh LipidContent CloneContent Clone20Countvarying lipid content of113different algae clonesCount10 0113380.2µm10150Count1.3µm10 3102150CountSide Scatter (SSC-HLog)10 4110102ChlorophyllA (RED-HLog)ChlorophyllA (RED-HLog)B. GatedbyChlorophyllA APlot 4:byGatedby ChlorophyllPlot 4: GatedChlorophyllA104CountSide-Scatter (SSC-HLog)10 5Plot 4: No GatedPlot 4: No Gated1041040100101102BODIPY Fluorescence103104104