T H E P O W E R O F E A S Y & A F F O R D A B L E F L O W C Y T O M E T R Y & C E L L C O U N T I N G

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1 T H E P O W E R O F E A S Y & A F F O R D A B L E F L O W C Y T O M E T R Y & C E L L C O U N T I N G

2 T H E P O W E R O F E A S Y & A F F O R D A B L E F L O W C Y T O M E T R Y & C E L L C O U N T I N G Many assays. One platform. Direct, absolute cell counts and viability Apoptosis Annexin V TUNEL Pan caspase Caspases 3/7 and 8 Mitochondrial membrane potential GFP expression Cell cycle Antigen detection Cell toxicity Cell proliferation Cell painting Cell signaling Antibody quantification Multiple cell types. Transformed Mammalian Primary Insect Suspension Yeast Adherent Beads. The desire to gain a systems perspective by analyzing whole cells has fueled a rising interest in cell- and bead-based applications for basic, applied, and clinical life science research, including drug discovery and development efforts. By studying intact cells, researchers can gain critical insights into the interactions among biological pathways. Further, they can leverage cell studies to expedite target validation, lead optimization, process development, toxicity determinations, clinical trials, and much more. Accelerate discovery and increase productivity. Guava Assays can be applied to a wide and ever-expanding range of applications. Cell counts and viability Cytotoxicity screening Cell culture optimization Bioprocess monitoring Apoptosis Cell line characterization Compound screening Bioprocess monitoring Antigen detection Compound profiling Protein/antigen expression analysis Hybridoma screening CD marker detection Cytokine quantification Cell cycle Cell line characterization Compound screening Bioprocess monitoring Cell tracking In vitro and in vivo partitioning Cell toxicity Cell proliferation Fluorescent proteins GFP screening Combined transfection efficiency and viability Bead-based assays Antibody quantification 2

3 [Guava Systems] have great value not only for assay development and drug lead optimization, but also as credible screening instruments giving high content readouts from a single well. Jill Gregory, Staff Scientist, Genzyme Corporation Drug Discovery Flow cytometry on demand. The Guava PCA and PCA-96 Systems as well as the Guava EasyCyte Mini and Plus Systems have been designed to meet the needs of today s life scientist. These on-demand systems, together with an ever-growing suite of assays, deliver powerful solutions for counting, profiling, and screening a most valuable commodityyour samples. Whether you focus on scientific applications, routine screening, cell counting, or commercial production, Guava Technologies has the solution that is right for you. Turnkey solutions. Guava Technologies has streamlined and simplified fluorescent cell- and bead-based assays by integrating instrument, software, and reagents into targeted, turnkey solutions. Samples in. Answers out. Next study. The assay development has been done for you, so you can concentrate on getting results. Four systems meet a variety of needs. Do more. Each Guava Assay meets exacting standards of robustness, ease-of-use, and affordability, while requiring only a few microliters of sample. And, Guava Systems are designed to be very easy to use, so you can be up and running within a few hours! Sophisticated cell analysis has never been easier. Do it right. Guava Systems combine fluorescence-based techniques and high quality optical elements to maximize assay sensitivity. And, automated software routines reduce operator-induced variability. The result? Accuracy and reproducibility. Do it fast. With an integrated system of instrument, software, and reagents from Guava Technologies, you don t have to rely on core labs to run your sophisticated cell analysis assays any longer. You can conduct your own studies in your own lab on your own schedule. PCA PCA-96 EasyCyte Mini EasyCyte Plus Laser Green Green Blue Blue Tiny footprint Guava PCA System Guava PCA-96 System Guava EasyCyte Mini System i n t u i t i v e s o f t wa r e i n t e r fac e G r e e n l a s e r M I N I M A L WA S T E M i c r o p l at e or tubes O P T I O N A L : - s i d e s c at t e r SCATTER Forward (FSC) Side (SSC) optional optional B LU E l a s e r Tube sampling Automated 96-well microplate sampling Small sample size (<20 µl) Minimal waste (<50 ml per day) Guava EasyCyte Plus System O P T I O N A L : - s i d e s c at t e r - 4 t h co lo r d e t e c to r FLUORESCENCE DETECTORS Green Yellow Red Near infrared optional 3

4 0 THE POWER OF CHOICE. MANY ASSAYS. ONE PLATFORM. Culture cells Guava Technologies has streamlined and simplified cell- and bead-based assays by integrating instruments and software with optimized reagents. Now, you can conduct cell analysis studies from your bench top, on your schedule. Now, you have the ability to generate data wherever and whenever you want to make better, faster decisions and maximize your laboratory s productivity. Guava Cell Cycle Assay Endpoint analysis Cell cycle. Determining the distribution of cells across the phases of cellular replication and division is done easily with the Guava Cell Cycle Assay. In addition to providing G0/G1, S, and G2/M phase percentages, this assay exports data in industry standard formats to allow more detailed analysis. M Cell Count S G2 G0 G1 Induce Split cells & select desired assays Guava Caspase 3/7 Apoptosis Assay Guava Caspase 8 Apoptosis Assay Guava Multi-Caspase Apoptosis Assay Guava Nexin Annexin V Apoptosis Assay Guava Mitochondrial Depolarization Apoptosis Assay Guava TUNEL Apoptosis Assay Cell counting and viability. Speed, ease, and depth: Unlike other viability assays, the Guava ViaCount Assay not only detects viable and dead cells, but also simultaneously assesses the apoptotic cell fraction. requiring only a single reagent, it is a simple nowash, mix-and-read assay that rivals the fastest of similar assays. Moreover, its versatility extends its applicability across a wide range of cell types, including suspended and adherent, mammalian and non-mammalian, and many others. Mechanism of action The Guava EasyCyte Plus System offers a convenient platform for exploring apoptosis biology, while addressing important applications such as endpoint analysis and mechanism of action studies. Based on the detection of fluorescent DNAbinding dyes, the ViaCount Assay offers far greater accuracy and reproducibility than traditional Trypan Blue-based methods, while using a much smaller cell sample. Because it can identify both viable and apoptotic cells, the ViaCount Assay can be used for cytotoxicity screens, proliferation assays, and cell culture monitoring. In addition, ViaCount reagents can assess cell health in any cell-based assay DNA Content (PM2) Multicolor antigen detection. Guava Express s Cell Surface Antibodies are fluorophore-conjugated antibodies that have been optimized for use on most Guava Systems. The antibodies provide quick, easy staining of human CD markers and allow for rapid multicolor immunophenotyping of hematopoetic cells. The Guava Express Assay and Guava Express Plus and Pro Software Modules allow you to use your own primary conjugates or our second-step reagents to detect surface and internal receptors or antigens on single cells. This is ideal for hybridomascreening, immunophenotyping, and other 2-, 3-, or 4-color assays. Accuracy Reproducibility 10e4 Observed Viability 100% 95% 90% 85% Guava ViaCount Trypan Blue/ Hemacytometer 80% 80% 85% 90% 95% 100% Expected Viability Low Viability Low Cell Density Guava ViaCount Assay Trypan Blue Guava ViaCount Assay Trypan Blue Percent CV CD45RA-PE (YLW-HLog) 10e3 10e2 10e1 10e0 10e0 10e1 10e2 10e3 CD3-PECy5 (RED-HLog) 10e4 4 At lower viability levels, the Guava ViaCount Assay provides more accurate assessments of cell culture viability than Trypan Blue dye exclusion assays using a hemacytometer. As shown above, the Guava ViaCount Assay provides superior reproducibility over manual cell counting with Trypan Blue in cell cultures with low viabilities or low cell concentrations. Dual staining of lysed whole blood with anti-cd45ra-pe and anti-cd3-pecy5 identifies naïve T cells (CD3+CD45rA+), memory T cells (CD3+CD45rA-), and B cells (CD3-CD45rA+). Cells in the lymphocyte gate were used for this analysis.

5 The Guava PCA is a major advance in our ability to rapidly and easily measure most of the parameters important in cell biology. Jennie Mather, CEO and President, Raven Biotechnologies, Inc. Cancer Therapeutics Apoptosis. In a process as complex as apoptosis, multiple methods of assessment are essential to identify cell populations in various stages of cell death. The Guava Apoptosis Assay Suite contains several complementary assays that can all be done on a single platform, quickly and easily. Caspases. By virtue of fluorochromeconjugated caspase inhibitors in combination with a viability dye, Guava Caspase Assays detect the activation of various caspases during the apoptotic process. Up to four separate populations (viable, early-to-mid apoptotic, late apoptotic, and dead) can be resolved in a single assay. These assays detect simultaneous activation of multiple caspases, caspase 3/7, or caspase 8. Mitochondrial membrane potential. Designed to save time and effort, the Guava Mitochondrial Depolarization Assay is a nowash, mix-and-read assay to monitor changes in mitochondrial membrane potential ( ψ), the loss of which is often associated with early apoptosis. Collapse of this potential may coincide with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol which triggers downstream events in the apoptotic cascade. Addition of the dead cell dye, 7 AAD, allows you to easily discern healthy, polarized cells from the apoptotic/depolarized and dead cells. Annexin V. Designed to save time and effort, the Guava Nexin Assay is a no-wash, mixand-read assay to identify cells in early- to midstage apoptosis. It is one of the fastest and easiest apoptosis assays available. Using Annexin V conjugated to a fluorophore, this assay detects the transfer of phosphatidyl serine from the internal to the external face of the plasma membrane during the initial stages of apoptosis. Addition of the dead cell dye, 7 AAD, allows you to easily discern early apoptotic cells from late apoptotic /dead cells. Induction of Early to Mid-Apoptotic Cells % Annexin V PE Azacytidine Ellagic Acid Mebendazole Albendazole Helenine Compound Concentration (µm) Compound dose response curve on Jurkat cells. Jurkat cells were treated with the indicated compounds at a range of concentrations for 24 hours before testing for apoptosis induction using the Guava Nexin Assay. Cell Count e0 10e1 10e2 10e3 DNA Strand Breaks (PM1) Up to four cell populations (viable, early-to-mid apoptotic, late apoptotic, and dead) can be detected using tilting markers in the Guava Multi-Caspase Assay. TUNEL. This classic assay for apoptosis-induced DNA fragmentation has been reworked to make it more convenient and accessible. The Guava TUNEL Assay detects late-stage apoptotic cells by using sensitive fluorescent probes to detect DNA strand breaks. The Guava TUNEL Assay easily distinguishes and quantifies the apoptotic cell population, as seen in this sample of HL60 cells treated with 20 µm camptothecin for 4 hours. 10e Fluorescence Emission Green JC-1 Monomers Orange JC-1 Aggregates Wavelength (nm) The Guava Mitochondrial Depolarization Assay can distinguish depolarized Jurkat cells, which fluoresce mostly green, from healthy uninduced cells, which fluoresce mostly orange (overlay). Fluorescent proteins. Screening clones of Green Fluorescent Protein (GFP) transfectants is now quick and easy: Guava Express Plus or Pro Assays automate single cell GFP detection in 96-well plates and combine it with viability assessment, apoptosis, or detection of a second (Plus) or third (Pro) marker. The Guava Express Plus or Pro Assays eliminate the need to test each clone individually since an entire plate of transfectants can be analyzed in a single experiment. Guava Express Plus & Pro Assays make screening for Green Fluorescent Protein (GFP) and other fluorescent proteins fast and easy. 5

6 The beauty of the Guava PCA System is that it generates reliable and reproducible quantification of apoptotic cells. Maureen E. Murphy, Ph.D., Department of Pharmacology, Fox Chase Cancer Center Cancer Research Cell tracking. Improve your cell tracking with three easy-touse, non-radioactive assays that are superior to population-based methods. Cell tracking assays from Guava Technologies use a well-characterized cell tracking dye, CFSE, that diffuses freely into cells and is retained within the cell without affecting cellular function. Cytotoxicity. When CFSE is used in combination with the dead cell dye, 7-AAD, all of the commonly performed cytotoxicity effector assays (NK, CMC, ADCC) can be performed with the Guava Cell Toxicity Assay. Cell proliferation. The Guava Cell Growth Assay is used to monitor cell proliferation of primary cultures out to five generations as well as to discern which portion of the cells are dead when stained with propidium iodide. Guava Cell Growth Assay Guava Cell Toxicity Assay Enriched T cells stimlulated with 5 µg/ml PHA, assayed on day 4 and analyzed using the ModFit software package from Verity Software House. Cell painting. The Guava Cell Paint Assay utilizes the cell tracking dye as a single color assay to track target cells within a mixed population for in vivo and in vitro applications. A second, user-defined dye can be added to perform a dual color assay for monitoring protein expression or antigen specificity. Hybridoma screening. Cytotoxicity data are automatically analyzed and displayed in an easy-to-read format. Using quantitative bead-based measurements, Guava RapidQuant IgG Assays quantify the levels of soluble IgG in hybridoma supernatants with much less work than traditional assays, such as ELISAs. Designed to be easy and effortless, rapidquant IgG Assays feature nowash protocols, superb day-to-day reproducibility, shorter time to answer, and improved accuracy over other available assays. Both mouse and human rapidquant IgG Assays are universalthey quantify all IgG subtypes of that species. Further, rapidquant Assays can be combined with other assays from Guava Technologies to accelerate hybridoma screening and monoclonal antibody development by allowing all stages of testing to be performed from less than 50 µl of supernatant. Optimized reagents. Optimized for use on all Guava Systems, reagents from Guava Technologies are formulated to ensure excellent sensitivity and ease-of-use. These unique reagents can also improve and accelerate your assays by eliminating many of the common preparation steps required with traditional protocols. In some cases, samples can be read immediately after staining, with no loss of sensitivity. Guava Express Pro Software. Accurate Prediction of Human IgG Concentrations Observed Concentration (µg/ml) Guava ELISA Expected Conc Expected Concentration (µg/ml), A280 Dilutions of various antibodies tested with the Guava rapidquant Human IgG Assay show excellent correlation with predicted values from ELISAs. Dedicated software. The friendly and intuitive Guava CytoSoft Operating Environment and Guava Assay-Specific Software Modules guide you through setup, maintenance, cleaning, and shutdown procedures as well as data acquisition and analysis. All modules are designed to streamline the experimental process from hypothesis to conclusion. results can be exported to spreadsheets or as industry-standard FCS 2.0 or 3.0 files for further analysis by third-party software. And, all software modules support compliance with 21 CFR Part 11 regulations. For multicolor detection, the Guava Express Pro Software Module allows you to view and analyze up to eight user-selectable histogram or dot plots simultaneously. You can also customize axis labels and statistics, and select multiple marker and gate types. Additional features include histogram overlays, backgating, zoom-to-plot for enlarged views, and batch-file printing. 6

7 T H E P O W E R O F S M A L L. T H E G U A V A F O R M U L A. Turnkey solutions for cell analysis from Guava Technologies are based on a superior interplay among reagents, software, and instrument. This sophistication elevates cell analysis and counting to an artwhile bringing it down to earth. The result: Tools that are truly a pleasure to use. The technology behind it all. So how does so much power and functionality get packed into such a small space? Credit a unique design that optimizes every available bit of space and eliminates wasted corners. Then, credit a unique microcapillary flow cell technology that eliminates the requirement for complicated sheath flow fluidics. The end result? An extremely compact, easy to use, and easy to maintain system, supporting on-demand cell analysis. Sheathless fluidics translates into fewer cells (<20 µl), less reagent, and minimal waste (<50 ml per 8 hours), saving you both time and money. Also, since the flow cell is self-aligning and user-replaceable, you can remove it at any time for cleaning and maintenance yourselfno more expense or downtime for service visits. And, of course, the tiny footprint fits into the tightest spots, reserving valuable laboratory bench space for other equipment and uses. The flow cell is easily removed for routine maintenance. Guava PCA System p h oto m u lt i p l i e r s ( P MT ) d e t e c t f lu o r e s e c e n t ly l a b e l e d c e l l s Guava EasyCyte Plus System SIDE-scatter (ssc) p h oto M U LT I P L I E R ( P MT ) D i f f e r e n t i at e s c e l l s b y G R A N U L A R I T Y F o r wa r d s c at t e r ( F S C ) p h oto d i o d e D i f f e r e n t i at e s c e l l s b y s i z e G R E E N l a s e r e x c i t e s f lu o r e s c e n t ly l a b e l e d c e l l s At the heart of Guava Systems is a 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. A green or blue diode laser excites the cellseach cell emits signals that are individually detected by photomultipliers and a photo diode. Guava Software Modules show all relevant data and results immediately. b lu e l a s e r e x c i t e s f lu o r e s c e n t ly l a b e l e d c e l l s 7

8 Expert support and service. All products from Guava Technologies are designed to increase productivity, maximize uptime, and minimize maintenance. But, in the rare instance when you do need assistance, our customer solutions team is always available to answer questions about our software, instruments, reagents, and applications. Individual members of the team specialize in different disciplines of life science research to ensure your questions are answered quickly, thoroughly, and completely the first time you call. Get started today. Discover how products and services from Guava Technologies can accelerate discovery and increase the productivity of your organization. For more information, contact us directly or visit: Specifications PCA PCA-96 EasyCyte Mini EasyCyte Plus Excitation 532 nm 532 nm 488 nm 488 nm Emission 580/20 nm 675/20 nm 580/20 nm 675/20 nm 525/30 nm 583/26 nm 680/30 nm Light scatter Forward Forward Forward Side (optional) Dimensions Weight 8.5 in (21.6 cm) h 12.6 in (32 cm) w 14.3 in (36.3 cm) d 32 lbs (13.2 kg) w/laptop 13 in (33 cm) h 16 in (40.6 cm) w 22.5 in (57.2 cm) d 80 lbs (35.6 kg) w/laptop Sample format 1.2 & 1.5 ml Tubes 0.5 ml Tubes 96-Well Plates Applications 9 in (23 cm) h 15.5 in (39.4 cm) w 17.9 in (45.5 cm) d 50 lbs (22.7 kg) w/out monitor 525/30 nm 583/26 nm 680/30 nm 785/70 nm (optional) Forward Side (optional) 13 in (33 cm) h 16 in (40.6 cm) w 22.5 in (57.2 cm) d 80 lbs (35.6 kg) w/laptop 1.2 & 1.5 ml Tubes 0.5 ml Tubes 96-Well Plates Specialty Guava ViaCount Assay Cell counting and viability Guava Cell Cycle Assay G0/G1, S, G2/M phase distribution Guava Express Cell Surface Antibodies 1 Antigen detection 2 2 Guava RapidQuant IgG Assays 1 Mouse & human IgG quantification n/a n/a n/a Apoptosis Guava Nexin Assay Annexin V binding Guava Caspase 3/7 Assay 1 Caspase 3/7 activation n/a n/a 3 Guava Caspase 8 Assay 1 Caspase 8 Activation n/a n/a 3 Guava Multi-Caspase Assay Pan caspase Guava TUNEL Assay DNA fragmentation Guava Mitochondrial Depolarization Assay Mitochondrial membrane potential n/a n/a Cell tracking Guava Cell Toxicity Assay Cytotoxicity: NK, CMC, ADCC Guava Cell Paint Assay Target cell monitoring Guava Cell Growth Assay Cell proliferation: Primary cultures n/a n/a 1 Requires Guava Express, Express Plus, or Express Pro Software Modules. 2 Users must optimize Guava Express Cell Surface Antibodies for the PCA and PCA-96 Systems. 3 Users may transfer prepared samples to tubes for collection on the EasyCyte Mini System. Optional software Guava Express Software Module (2-color) Antigen detection; immmunophenotyping n/a n/a Guava Express Plus Software Module (3-color) Antigen detection, GFP, immunophenotyping, antibody quantification Guava Express Pro Software Module (4-color) Antigen detection, GFP, immunophenotyping, antibody quantification, cell signaling n/a n/a n/a n/a n/a Global Headquarters Guava Technologies, Inc Industrial Blvd. Hayward, CA USA Tel: Fax: info@guavatechnologies.com Europe & Africa Guava Technologies, Inc. 3 Southview Business Centre Tinwell Road Stamford, Lincs PE9 2JL UK Tel: +44 (0) Fax: +44 (0) sales@guavatechnologies.com Asia-Pacific Guava Technologies Inc. 1502, DLF Galleria, DLF Phase 4 Gurgaon Haryana, India Tel: Fax: info-ap@guavatechnologies.com For Research Use Only. Not for use in diagnostic procedures. ModFit is a trademark of Verity Software House. Guava, Guava Technologies, the Guava Logo and all other trademarks are the property of Guava Technologies, Inc Guava Technologies, Inc. All rights reserved. MK Mar07 5K