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Data Sheetguava easyCyte Flow Cytometry SystemsFlexible, Intuitive and AffordableThe guava easyCyte flow cytometrysystems are easy to use and delivercomplete and comprehensive cellanalysis—right on your benchtop. Theculmination of over a decade of flowcytometry expertise, these instrumentsconsume less sample, generate lesswaste and are easier to use and maintainthan traditional flow cytometers—allwhile providing the analytical power youneed to expand your research horizons.Single blue (488 nm), dual blue and red (642 nm),or triple blue, red, and violet (405 nm) excitationlasers provide up to 12 simultaneous detectionparameters, including 10 fluorescent colors plusforward and side scatter for size and granularitydetermination. The guava easyCyte family alsomeets your sample throughput needs by offeringboth single sample and multi-sample processing.The guava easyCyte HT instruments providehigh throughput analysis with a robotic sampletray that automatically handles a 96-well microplate and up to 10 sample tubes, while theguava easyCyte systems enable single sampleprocessing with additional cost savings.Benefits Flexible ― Up to 12 detection parameters― Multi-sample or single sample processing Easy to Use― No sheath fluid required, enabling small samples and low waste― Intuitive software― Absolute cell counts—determine accurate cell numbers andpopulations without reference beads A ffordable― Designed and priced for every laboratory and budgetMerck Millipore is a division of

Microcapillary Flow CytometryGuava -Patented microcapillary systemAt the heart of every guava easyCyte system isa patented, microcapillary flow cell that eliminatesthe need for sheath fluid and provides absolutecell counts. This translates into less complexity,smaller samples and minimal waste, saving youboth time and money. Plus, the flow cell is selfaligning and user-replaceable to reduce downtimeand service visits.Sheath Fluid:NoneMicrocapillaryWaste: 50 mL/8 hour runLaserSample Flow Typical # Cells Per Protocol:1,000 – 10,000 cells/test No laser alignment or sheath fluid required Usessmaller sample volume and generates lesswasteWasteFlow CellSampleTraditional sheath fluid system Aspirates sample directly from the tubeWaste:8,000 mL per 8 hour runTraditionalWasteTypical # Cells Per Protocol:100,000 – 1,000,000 cells/testSheath Flow LaserInjection TipSheath Fluid: 10 mL/testSample Flow Flow cell is user replaceable for minimaldowntimeInside the guava easyCyte 12HT and guava easyCyte 12 systemsSee page 7 for specifications.How it WorksThe guava easyCyte systems use patented, microcapillary, laser-based technology capable of detecting mammalian and microbial cells and beads. A sample of fluorescently labeled cells is aspirated into a uniquely proportioned microcapillary flow cell. Ared, blue or violet laser excites fluorophores, and each cell emits signals that are individually detected by photomultipliers 50RED695/50YEL583/26FSCSSC405 nm488 nm642 nm2

101102Plot P03, Gated on P01.R1Count0 40 80 120 160 200 240Plot P02, Gated on P01.R1100103104105100101Green-B Fluorescence AreaPlot P04, Gated on P01.R1100101102103102103104105Yellow-B Fluorescence Area104105Count0 40 80 120 160 200 240Count0 40 80 120 160 200 240The guava easyCyte flow cytometers use up tothree lasers (488, 642, and 405 nm), to achieveup to 12 simultaneous detection parameters,including 10 fluorescent colors plus forward andside scatter. In the two- and three-laser systems,the lasers overlap spatially and are modulatedout of phase at high frequency so that each particle is sampled many times as it travels throughthe overlapped beams. Modulation is particularlyimportant for identifying dyes which have overlapping emissions, such PE-Cy7 (blue laserexcitation) and APC-Cy7 (red laser excitation).Unlike spatially separated beams, modulationeliminates the need for time-delay calibration, simplifying the overall operation of the instrument.Count0 40 80 120 160 200 240Three-Laser ExcitationPlot P06, Gated on P01.R1100101102103104105Blue-V Fluorescence AreaRed-B Fluorescence Area Up to 12-parameter analysis (10 colors, plusforward and side scatter for size and granularitydetermination) Compatible with commonly used fluorophoresand dyes Eliminates the need for time-delay calibrationImmunological PhenotypingCD3-Plot P03, gated on PO1.CD45-.CD3 101102103CD16 CD56 PE (YEL-B-HLog)CD8 CD62L FITC (GRN-B-HLog)101102103CD4 PE-Cy7 (NIR-B-HLog)102103CD16&CD56 101CD19 101102103CD45RA APC (RED-R-HLog)104Plot P06, gated on P01.CD45 .CD3 .CD104104CD19 BV 510 (GRN-V-HLog)101102103CD4 R6CD3 Plot P04, gated on PO1.CD45 .CD3-.R9100CD62L FITC (GRN-B-HLog)101102103102103101CD3 BV 421 (BLU-V-HLog)100CD3 CD4 CD8 101102103CD8 APC-Cy7 (NIR-R-HLog)104CD45 102103101CD45 PerCP-Cy5.5 (RED-HLog)CD3-104100CD45 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, ungatedR6101102103CD45RA APC (RED-R-HLog)10410 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-PE, CD56-PE, anti-CD19 Brilliant Violet 510, anti-CD45 RA APC, and anti-CD62L FITC. After incubation, cellswere 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. Lymphocyte cells (CD45 ) were gated into a SSC vs. CD3 plot. T cells (CD3 and CD45 ) were gated into a plot comparing CD4 and CD8 positive cells. CD4and CD8 naïve memory cells were further discriminated by evaluating each population in a CD45RA (naïve T cells) vs CD62L (memory T cells). To separate thenatural killer (NK) and B cells from the lymphocytes, CD3-negative cells were gated into a plot comparing CD19 (B-cells) and CD16 56 (NK cells). As the figureshows, separation is visible and is comparable to other published data.3

SoftwareThe guavaSoft operating system software provides access to modules for acquisition and analysis, as well as instrument setupand maintenance. The guavaSoft operating system includes templates for use with a wide range of Merck Millipore flowcytometry kits to simplify your experiments and data collection. Additionally, the guavaSoft package includes InCyte software,an intuitive open software package for analysis. Results can be exported to spreadsheets or as industry-standard FCS 2.0 or 3.0files for further analysis. GuavaSoft software has 21CFR Part 11 enabling features.InCyte Software: IntuitiveMerck Millipore’s InCyte software has an intuitive, easy-to-use interface that enables you to focus on data at either the sample or experimental level. The software simplifies setup and analysis of plots with the drag-and-drop features, while automatedcompensation makes it easy to perform complex, multi-color assays. The instant update feature responds in real time to changeanalysis conditions for viewing. InCyte software allows you to analyze entire plates of data in the time it would previouslyrequire to analyze a single sample using the multi-parameter heat mapping function. These features provide a simple and rapidmeans 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.Perform compensationduring acquisition or analysisor use the automatedcompensation features.View up to 24 plots at once.Drag and drop gating.Real-time adjustment.Plot PO2, gated on P01.R1MitoSense Red ( )Annexin V ( )4.44%Default orcustom statistics.10310140001026000Side Scatter (SSC - HLin)2000110110210103Forward Scatter (FSC - HLog)2345678MitoSense Red (-)Annexin V (-)6.19%10010110010049A101112102MitoSense Red (-)Annexin V ( )20.90%103104Annexin V CF488 (GRN - HLog1040Create and applyanalysis methods acrossmultiple datasets.MitoSense Red ( )Annexin V (-)68.46%MitoSense Red (RED2 - HLog)8000104Count: 2746Plot PO1, ungatedMultiple plot andgating options.Plot PO3, gated on P01.R17AAD (RED - HLog)103BC102DE101FMinimal gain adjustmentneeded when performingroutine assays.100GH100Allows for analysis ofboth tubes and plates.4101102103Annexin V CF400 (GRN - HLog104

InCyte Software Heat Map ViewHeLa 24 hours123456789101112Combine groups of data toconstruct heat maps, IC50,or EC50 pase3/77-AADEFG020%20 40%40 60%60 80%80 100%HHeLa cells in microtiter plates were treated with multiple cytotoxic compounds for 24 hours. Cells were stained using Merck Millipore’s MitoDamage, MitoCaspase,or MitoStress kits. Cells were analyzed on the guava easyCyte system and percent population data were compared in a heat map format using MerckMillipore’s InCyte Software. InCyte software allowed for the quick identification of hit compounds and comparison of all 5 parameters simultaneously asshown in the pie charts above. The data above shows the data for the 80 compounds on both cell lines.Response Level, %0 10 20 30 40 50 60 70 80 90 110IC50 Determination within InCyte SoftwareResponse Level, %0 10 20 30 40 50 60 70 80 90 11010-110-1IC50 38µM100101102103101102103ConcentrationIC50 4.9µM100ConcentrationIC50 determination using the Cytochrome c Kit and analyzed the built-in IC50/EC50 curve fitting feature of InCyte software. Cells were analyzed on the guavaeasyCyte 8HT system. Plot A shows the drag and drop gating strategy used for the IC50 determination. Plot B shows the IC50 curve results for gambogic acidand Plot C shows the EC50 for etoposide. The once-complex task of finding the IC50 or EC50 for a compound has been made as easy as a few clicks of the mouse.5

Anatomy of the guava easyCyte Systemsguava easyCyte Single Sample System2.6.1.4.5.3.Number1.2.3.4.5.6.7.System Feature DescriptionUp to 12 simultaneous detection parametersMicrocapillary flow cellWash vialWaste vialSmall footprint saves valuable laboratory spaceSingle sample loaderRobotic sample tray for 96-well microplateguava easyCyte HT System62.3.1.4.5.7.

guava easyCyte System FeatureseasyCyte easyCyte easyCyte easyCyte easyCyte easyCyte easyCyte easyCyte 55HT6-2L6HT-2L88HT1212HT0500-5005 0500-4005 0500-5007 0500-4007 0500-5008 0500-4008 0500-5012 0500-4012SystemCatalogue No.Violet (405 nm) LaserBlue (488 nm) Laser Red (642 nm) Laser FSC SSC Blue-V (448/50 nm) Green-V (525/30 nm) Yellow-V (583/26 nm) Red-V (695/50 nm) Green-B (525/30 nm) Yellow-B (583/26 nm) Red-B (695/50 nm) NIR-B (785/70 nm)Red-R (661/15 nm)NIR-R (785/70 nm) Microcapillary Fluidics Direct, Absolute Cell Counts Automation-plate and tubes Mixing Dell Laptop InCyte Software Digital Signal Processing Spectral Bands and Applicable DyesFor a complete list please see: www.merckmillipore.com/guavaBlue (448/50 nm)DAPIHoescht 33258Alexa Fluor 405Marnia Blue dyePacific Blue dyeCascade Blue dyeLIVE/DEAD VioletDyLight 405eFluor 450Zombie Aqua dyeBrilliant Violet dyeGreen (525/30 nm)Alexa Fluor 430Pacific GreenBrilliant Violet dyeQdot 525Qdot 545FITCGFPAlexa Fluor 488CF488FAMQdot 525Acridine OrangeSYBR GreenSYTOX Green JC-1BODIPY-FLCalceinCFSEOregon Green dyeYellow (583/26 nm)Pacific Orange dyeBrilliant Violet dyeQdot 565Qdot 585Alexa 555Alexa 568CF555PE-B, PE-RQdot 565Acridine OrangedsREDEthidium BromideSYBR GreenSYTOX OrangeJC-1TMRETRMRCFSENile RedRed (661/15 nm)Alexa Fluor 647APCCD647Cy5Qdot 655DRAQ5Ethidium BromideEthidium HomodimerSYTOX RedTO-PRO -3TOTO-3DilC1(5)MitoSense RedBODIPY 650/665405 nm Laser488 nm Laser642 nm LaserRed (695/50 nm)eFluor 650Brilliant Violet dyeQdot 7057-AADPropidium IodidePE-Alexa Fluor 647PE-Alexa Fluor 700PE-Cy5PE-Cy5.5PE-Texas Red dyePerCPPerCP-Cy5.5Qdot 705DRAQ5Ethidium BromideEthidium HomodimerLDS-751Nile RedNIR (785/70 nm)PE-Alexa Fluor 750Propidium IodidePE-Cy-7APC-Cy7APC-Alexa Fluor 7507