DS-9819A Insect Cell Analyses Using the Beckman Coulter ® Vi-CELL™ VI-CELL XR™ Part of the Cell Lab family Instrument Settings When using the Vi-CELL most cells can be analyzed using the default instrument settings. In select cases, certain cell types may require modification of the default analyses parameters. The ViCELL software allows for easy user selection of the optimum parameters thus ensuring accurate results. The Vi-CELL settings for SF9 insect cells are the following: Insect Cell Analyses Insect cells are used to produce therapeutic proteins. The specific gene coding the protein of interest is introduced into the cell by a DNA virus of the family Baculoviridae. Protein production via this mechanism, virus infection of the insect cell, is referred to as the Baculovirus Vector System. The most common insect cell used is the SF9 cell or Spodoptera frugiperda species. Equipment Used Beckman Coulter Vi-CELL XR Validated Vi-CELL XR reagent pack Min. Size (µm) = 9 Cell Brightness = 85 Max. Size (µm) = 50 Cell Sharpness = 100 Number of Images = 50 Viable Cell Spot Brightness = 75 Aspirate Cycle = 1 Min. Circularity = 0 Trypan Blue Mixing Cycle = 2 Decluster Degree = Low Results The results reported by the Vi-CELL are shown in the Results Section of Figure 1. Percent viability and concentration were 95.1% and 2.01 x 106/ml. The Vi-CELL also reports viable cell concentration, mean cell diameter, and cell circularity. Graphs may be expanded via a mouse click. Real time cell images may be archived for future re-analyses. Conclusion The Beckman Coulter Vi-CELL automates the standard manual method, providing accurate results and removing the subjective nature inherent in the manual method. REAL TIME CELL IMAGES: (UP TO 100 IMAGES MAY BE VIEWED) PERCENT VIABILITY CELL CONCENTRATION SIZE DISTRIBUTION REAGENT LEVEL MONITOR Figure 1. Viable cells are circled in green and non-viable in red. THE VI-CELL MANUAL TRYPAN BLUE DYE EXCLUSION METHOD The Vi-CELL automates the widely accepted Trypan Blue Dye Exclusion method. The Vi-CELL combines the state of the art in imaging technology, proprietary algorithm and fluidics management. At the heart of the Vi-CELL is the customized liquid handling system. This system, which allows sample aspiration, reagent handling and subsequent instrument cleaning, is fully automated. Once the cellular suspension has been aspirated and mixed with the trypan blue dye, it is pumped to the flow cell for imaging. The Vi-CELL can analyze up to 100 images for a given analysis increasing total volume from 15 to 30 times over the manual method with result in a less that 2.5 minutes. • Automated liquid handling • Automated lysing • General purpose centrifugation • High performance centrifugation • Ultracentrifugation Prepare • Automated fluorescence microscopy • Cell counting • Cell markers • Cell viability analysis • Flow cytometry • Monoclonal antibodies • Automated liquid handling • Flow cytometry • Microarray technology • Monoclonal antibodies • Signal transduction assays Identify Probe Sort • Cell sorters • Micro-piezeo electric tips • Reagents (various) Evaluate • Monoclonal antibodies • Multi-mode plate reading • Genomics solutions • Proteomics solutions • Software informatics As mentioned, the standard method for measuring cell viability is the Trypan Blue Dye Exclusion method. Trypan blue stain (0.4%) is mixed with an equal volume of cells.Viable cells, given their intact membranes, exclude the trypan blue stain; non-viable cells, membrane permeable, stain dark blue. The manual method, however, requires a technician, using a hemacytometer and microscope, to enumerate both stained and unstained cells and manually calculate the percent viability. In addition to being labor intensive , this technique has substantial accuracy error due to it’s subjective nature. VI-CELL TECHNICAL SPECIFICATIONS INSTRUMENT FUNCTION: Concentration Range: 5 x 104 to 1 x 107 cells / mL *Counting Accuracy: + 6% OPERATING SYSTEM: Power 50 watts (65 Watts Max.) Voltages 100V, 120V, 220V or 240V 50/60 Hz Windows® 98 Windows® 2000 Windows® XP TEMPERATURE: INSTRUMENT TYPE: 11.3kg (25lb) 10° to 40° C (50° to 104° F) WEIGHT: Video imaging through a quartz flow cell • Automated liquid handling • Flow cytometry • Immunoassays • Monoclonal antibodies • Software algorithms Diagnose POWER REQUIREMENTS: UNIT DIMENSIONS: 44.5cm (17.5”) height 38cm (15”) width 41cm (16”) depth VI-CELL SERIES PN AUTO SAMPLE VI-CELL XR 383556 Yes SIZE RANGE (µm) 2-70 SAMPLE VOLUME ANALYSIS TIME (mL) (Min) 0.5 <2.5 VIABILITY RANGE 0-100 VI-CELL AS 6605769 Yes 5-70 1.0 <3.5 0-100 VI-CELL S 383080 No 5-70 1.0 <3.5 0-100 VI-CELL XR QUAD PACK VI-CELL AS, S QUAD PACK VI-CELL CONCENTRATION CONTROL VI-CELL FOCUS CONTROL 383722 IMAGING TECHNOLOGY Auto-focus routine Firewire Camera 1394 X 1040 CCD array Manual focus routine Image frame grabber 640 X 480 CCD array Manual focus routine Image frame grabber 640 X 480 CCD array 383198 175478 175474 Developing innovative solutions in Systems Biology. ® B2004-6327 ©2004 Beckman Coulter, Inc.
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