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Books Audiobooks Magazines Podcasts Sheet Music Documents (selected) Snapshots Guava EasyCyte System User's Guide Uploaded by Armando Signore 100 (1) 100 found this document useful (1 vote) 866 views 395 pages Document Information click to expand document information Description: Learn to use the flow cytometer Guava system.Cancel anytime. Share this document Share or Embed Document Sharing Options Share on Facebook, opens a new window Share on Twitter, opens a new window Share on LinkedIn, opens a new window Share with Email, opens mail client Copy Text Related Interests Cytotoxicity Apoptosis Flow Cytometry Technical Support Computing Footer menu Back to top About About Scribd Press Our blog Join our team. Quick navigation Home Books Audiobooks Documents, active. The easyCyte line of flow cytometers offers: For applications requiring exceptional sensitivity, a high-power blue laser (HPL) is offered in both single-loader and high-throughput (HT) formats. In addition, these simple fluidics enable a self-aligning flow cell that can be removed for cleaning by the user. New, more robust lasers available in virtually any combination mean that you choose the excitation and emission channels that are most relevant to your lab’s experiments. Each of the versatile optical configurations can be equipped with an affordable single-sample loader, or an integrated multi-well sample tray option, enabling worry-free walkaway acquisition of up to 96 samples. As a result, the instrument is very compact in size and simple to operate. This feature makes the easyCyte Systems beneficial for measuring a wide variety of cells and particles. A positive-displacement syringe pump enables not only direct sampling, but can adjust the flow rate as well to ensure clear data from a range of sample titers.
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To ensure that cells stay in suspension during sampling, a rotating, variable-speed mixing paddle with auto-cleaning is used between each well, so there is no need for manual resuspension after loading the plate. This virtually eliminates carryover between samples, and mixes more efficiently than shaking plate holder formats. The manual loader holds two separate tubes, allowing the user to progress quickly from one sample to another, or to wash between samples following an acquisition. This means that there is no user-adjustment necessary. The kit consists of easyCheck standardized fluorescent beads and diluent, facilitating the assessment of accurate counting and fluorescence detection. The easyCheck Kit is included with all instruments to enable daily routine signal optimization and quality control. Absolute counts can be done using cellular or bead samples. Linear and accurate results can be seen across the multiple concentrations and on a variety of cell and sample types. This increased resolution and sensitivity allows for better separation, making gating and identification of dim populations easier. These capabilities may prove particularly useful for researchers analyzing particulates, beads, bacteria, and algae.Sample concentrations were within the acceptable range for all samples within the plate. Each well was acquired and concentrations were determined.Data shown below confirms that carryover is minimized with this format. Because the systems are easy to use and affordable to operate, you can achieve more productive research. Moreover, you can now analyze a variety of cell types, from small bacteria to algae, yeast, plant, and mammalian cells. You can export to spreadsheets or utilize any third-party analysis format. 21 CFR part 11 enabling features are included.It has an intuitive, easy-to-use interface that makes it possible to visualize and compare up to eight data sets at the same time.
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The software is ideally suited for interrogation of high-content data sets derived from multiple functional studies, such as those commonly employed during compound screening and target identification. In addition, data acquisition is fully incorporated, so acquisition and analysis can be performed on the same instrument. Most importantly, comparative results are displayed at the experiment level, rather than on an individual well or sample basis. Cells were stained using Guava MitoDamage, MitoCaspase, or MitoStress kits. InCyte Software allowed for the quick identification of hit compounds, as well as the comparison of all 5 parameters simultaneously as shown in the pie charts above. The figure shows the data for the 80 compounds on both cell lines. Plot B shows the IC 50 curve results for gambogic acid, and Plot C shows the EC 50 for etoposide. The once complex task of finding the IC 50 or EC 50 for a compound is now performed with just a few clicks of your mouse. Optimized for each assay, Guava assay-specific software modules display only the plots and statistics you need. Each software module acquires and analyzes data, then exports results to a database without additional user intervention. This integrated, automated process makes your cell analysis both easier and more accurate. Their combined expertise enables them to answer questions beyond hardware and software, as well as to help address any cellular analysis challenges you face. If your concern cannot be addressed by our Technical Support Team on the telephone, we will schedule a visit from a Field Service Engineer or Field Applications Specialist. These highly qualified scientists have expertise in both flow cytometry and the entire Luminex portfolio of flow cytometers, kits, and reagents. Introductory (one half-day) and Advanced (one full-day) training visits are available, and can be tailored to the experience and needs of the laboratory.
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This provides added peace of mind that your instrument is producing high-quality data. A Luminex Field Service Engineer will come to your site to perform the testing and provide the necessary documentation for regulatory compliance. Moving a Guava easyCyte instrument without proper procedures could negatively impact system performance and void your one-year instrument warranty, per Luminex’s terms and conditions of sale. Simply select your instrument, configuration, and fluorochromes to view the excitation and emission spectra specific to your instrument. Standard Luminex policy requires instruments with a lapse in service coverage to be recertified by Luminex prior to commencing a service plan. The recertification fee includes travel and three hours of labor. Additional hours of labor and parts used during the recertification will be invoiced. Additional Services are not covered by any service plan. Labor and travel not included. Not for use in diagnostic procedures. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any form, or by any means: electronic, mechanical, magnetic, optical, or otherwise, without the prior written permission of EMD Millipore Corporation, Industrial Blvd., Hayward, CA 94545, United States of America. Disclaimer EMD Millipore reserves the right to change its products and services at any time to incorporate the latest technological developments. This guide is subject to change without notice. EMD Millipore has thoroughly tested the operation of guavasoft Software on Microsoft Windows XP and Windows 7 operating systems, but does not warrant that the software functions correctly on any other operating system. EMD Millipore has not validated the analysis of guava easycyte System data using thirdparty programs and cannot warrant that the results using these programs will be correct. EMD Millipore does not provide support for any third-party programs.
Limitations The guava easycyte System is for research use only; not for use in diagnostic procedures. The results of the assays are dependent upon the proper use of the reagents and instrument. Please refer to the appropriate package insert for specific instructions and limitations. Trademarks The M logo is a trademark of Merck KGaA, Darmstadt, Germany. Millipore, FlowCellect, InCyte, easycheck, guavasoft, ViaCount, Nexin, and guava are registered trademarks or trademarks of EMD Millipore Corporation. Microsoft, Windows, Internet Explorer, and Excel are registered trademarks of Microsoft Corporation. Dell is a trademark of the Dell Computer Corporation. All other trademarks are property of their respective companies. Patents The guava easycyte System is the subject of issued and pending US patents and foreign equivalents, including the following US patents: 5,798,222 Apparatus for monitoring substances in organisms 6,403,378 Cell Viability assay reagents 6,710,871 and 6,816,257 Method and apparatus for detecting microparticles in fluid samples 7,320,775 Exchangeable flow cell assembly with a suspended capillary 7,410,809 Particle or cell analyzer and method 8,184,271 and 7,847,923 Differentiation of flow cytometry pulses and applications Rev B December 8, 2014 The system is equipped with three lasers violet, blue, and red allowing you to analyze up to 10 fluorescence parameters, as well as forward and side scatter. It accommodates individual microcentrifuge sample tubes.Use the guava InCyte software module to acquire and analyze data. InCyte is an open software package, which you can also use to analyze FCS 3.0 data files acquired from any guava software module. For a list of assay-specific software modules, see Program Search List on page 1-9. guava InCyte application templates are available for all FlowCellect assays. The templates provide the plots, parameters, analysis tools (Methods), and statistics for each application.
For more information on these templates, refer to the specific reagent kit user guide. vii This guide is intended for the guava easycyte 5, 6-2L, 8, and 12 instruments. Any reference to NIR, Red2, and NIR2 parameters, may not apply to your specific instrument. Refer to easycyte Lasers and Fluorescent Filters in Specification for a complete list of the parameters available with each system. For information on using the other guava software modules, refer to the guavasoft Modules User Guide. This guide assumes you have a working knowledge of Microsoft Windows operating system. If you have any questions regarding the Dell computer or the operating system, refer to the appropriate manufacturer s documentation. For information on preparing samples, refer to the appropriate reagent kit user guide that was shipped with your reagents. Conventions Used in This Guide NOTE: Points out additional information that may be helpful. WARNING: Alerts you to situations that could result in bodily harm, instrument damage, failure in a procedure, or incorrect results. Help Safety Bold: Indicates buttons to click or options within the software to select. Italics: Used for names of user guides and package inserts, as well as messages that appear on the screen. 1 Read through the section of the guide specific to the operation you are performing. Refer to the table of contents and index to locate information. Use the system only as directed in this user guide. Do not perform instrument maintenance or service except as specifically stated. Please read the following safety information before using the system.Follow these guidelines: The use of tubes other than those specified may result in damage to the instrument. Do not run any other programs, including Internet Explorer, on the laptop while using guavasoft Software to acquire data.Running other programs during a run may interfere with acquisition or interrupt the run.
Biological Safety WARNING: All biological specimens and materials that come into contact with them can transmit potentially fatal disease. To prevent exposure to biohazardous agents, follow these guidelines: Handle all biological specimens and materials as if capable of transmitting infection. Dispose of waste using proper precautions and in accordance with local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. Add 10 ml of bleach to the waste vial after emptying it. Dispose of waste in accordance with federal, state, and local regulations. Electrical Safety The instrument must be connected per the instructions in the user guide. The power conditioner is required to meet electrical compliance. WARNING: Turn off the main power switch at the back of the instrument and disconnect the power cord before replacing fuses. For the actual power for a specified instrument model, see easycyte Lasers and Fluorescent Filters under Specifications. ix Light shields within the instrument enclose the path of laser radiation. Additionally, the instrument enclosure provides secondary protection from any laser radiation. WARNING: To avoid exposure to laser radiation or electric shock, follow these guidelines: Do not open the instrument or attempt to perform any internal maintenance. There are no user-serviceable parts. Turn off the power to the guava easycyte System before attempting to remove the flow cell. WARNING: The use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure Labels Following are examples of the labels affixed to the guava easycyte instrument: NOTE: The following caution label is visible when the flow cell access panel is removed. The panel is to be removed when the system is powered off. Even when powered on, the accessible laser radiation falls within Class I limits.
Refer to the appropriate reagent kit user guide for specific instructions and limitations.The system includes the easycyte instrument, a laptop computer with pre-installed software for data acquisition and analysis, and optimized reagents and protocols.In addition, the software includes the guava easycheck module, which verifies that the system is performing optimally, and Guava Clean, a cleaning module that allows you to clean the instrument s fluid system prior to shutdown.In addition, results are exported to a spreadsheet file.Place the instrument on a stable surface in a dedicated location in the laboratory. Allow at least 4 inches between the back of the instrument and the wall for proper ventilation. Maintain easy access to the power cord in case the instrument needs to be disconnected in an emergency. The initial installation will be performed by an EMD Millipore field service representative. WARNING: To avoid damage to the instrument, be sure to remove the shipping restraint before plugging in the instrument. NOTE: If the instrument needs to be moved to a new location in the lab area or building, always use two people to lift and a sturdy transport such as a cart. If a longer distance move requiring packup is required, contact EMD Millipore. A Relocation and Installation service is available for a fee.NOTE: The power conditioner is required to meet electrical compliance. 4 Connect the laptop power supply to the laptop. Plug the power supply into the power cord, then plug the power cord into the power conditioner. WARNING: The power conditioner is not a continuous power supply. Ensure that the instrument is powered on during acquisition. Connecting the guava easycyte System 5 You can connect the laptop to a local network or the internet. Contact your network administrator for assistance. You can also connect a printer. If you connect a printer, you must install the appropriate print drivers.
6 The easycyte System comes with two sets of sample tube holders: a set of holders for 1.5-mL microcentrifuge tubes and a set of holders for titer tubes. Select the set you want to use and install them by screwing each holder onto the loader assembly as illustrated below. Installing and changing the tube holders The easycyte System supports the following tubes: 1.2-mL microtiter tube with conical tip 1.5-mL microcentrifuge tube with conical tip and screw cap (if snap-cap tubes are used, cut off the cap) Hardware Connections 1-3 Follow the instructions in Running Guava Clean on page 3-2 using deionized water in place of cleaning solution. Keep the tube of water loaded on the system whether you are ready to run samples or are going to shut down the system.During start-up, laptop freezes on particular screen. Message: The instrument could not be detected. Message: The instrument appears to be either off or not connected. You can run in Analysis mode only.Laptop is set up for authorization. System may be searching for directory during startup. Communication problem. 1. guava easycyte System is not turned on or is not getting power. 2. Cable connection between easycyte System and laptop is loose. 3. easycyte System and laptop were not powered on in correct sequence or have lost communication. Registration code not entered or not entered correctly. Do not enter password. Click OK or Cancel to continue. Contact your IT department for assistance with any modifications. The original laptop setup does not require a password. Press Enter to continue. Reboot system, if necessary. If message appears after rebooting, contact EMD Millipore Technical Support. 1. Ensure easycyte System power cord is properly plugged in and system is turned on. 2. Ensure USB cable is securely connected to laptop. Use USB 2.0 ports if possible. Reboot computer, if necessary. 3.
Turn off easycyte System, exit guavasoft software, restart laptop, turn on easycyte System, wait for indicator light before starting the software. Enter registration code and ensure all characters are correct. 1-4 guava easycyte System User Guide Laptop keeps shutting down. For InCyte only Message: Sorry - this unlock is invalid. Loose power cable. 1. Power supply to laptop is faulty. 2. Screen saver is interfering. 3. Laptop overheating. Ensure instrument is plugged. Check all power connections. 1. Ensure laptop is plugged in correctly. Use surge protector and ensure it is plugged in and turned on. 2. Adjust power scheme screen saver options. Double-click Display, select Screen Saver tab, click Settings under Energy saving features of monitor. Make sure Setting for Always On power scheme are all set to Never. Laptop should not be allowed to sleep.Incorrect or no unlock key. Ensure the correct unlock key is entered. If necessary, contact EMD Millipore Technical Support to obtain unlock key. WARNING: Do not run Excel software, Internet Explorer browser, or any other program on the laptop while using guavasoft software to acquire data from the guava easycyte System.Running other programs (even if you are not actively using them) during a run may interfere with acquisition or interrupt the run.The power switch, sample loader, and waste and cleaning solution vials are the only instrument components that you will routinely handle, and most of your interaction is through the software via the laptop. The power switch is located on the back-right side of the instrument.The loader arm can be lowered and swiveled, allowing you to quickly switch from one sample to the next during acquisition. To load samples, pinch the release lever with your thumb and forefinger and gently lower the arm assembly (figure 1). Once lowered, rotate the assembly to change the sample tube (figure 2), then push up on the assembly until you hear a click.
For information on filling the vial, refer to Filling the Cleaning Solution Vial on page 3-4. The waste vial (located on the left ) captures the sample fluid after it exits the fluid system. Empty the waste vial at the end of each day or more often, if necessary. Add 5 ml of bleach to the vial after you empty it (see page 3-5 for information). Fluid System Sample uptake occurs through a capillary and is regulated by a variable-speed fluid pump. The pump does not require sheath fluid or other supplementary fluids for operation. Because the system s sampling precision depends on the integrity of the fluid pathway, it is important to maintain a clean system. Do not allow samples to remain in the capillary for extended periods of time. Perform frequent cleaning cycles to prevent the build-up of cellular debris that may restrict sample flow. Always keep a tube on water loaded when the instrument is not in use. If a clog does occur, you can clear it by using the backflush capillary feature, which reverses the flow of fluid and flushes it out of the flow cell at a high speed. Refer to Backflushing the Fluid System on page 3-4 for detailed instructions on using the backflush feature. Some assays allow you to select the sample flow rate for acquisition. For most assays, the Medium flow rate is the default. If the sample concentration is higher, dilute the sample or use a lower flow rate. For assays where the peak CV is critical, such as Cell Cycle, use the Low or Very Low flow rate. Always use the same flow rate for acquisition that you use for adjusting the settings. Laser The guava easycyte has three lasers blue, violet, and red. During acquisition using InCyte or easycheck, all three lasers turn on. During acquisition using ExpressPro, the blue and red lasers turn on. During acquisition using any of the other guava software modules, only the blue laser turns on. The lasers turn off when acquisition is complete. Software CD A CD contains guavasoft software.
The guava easycyte System User Guide is installed with the software.The guavasoft software main menu allows you to select an assay from either a list of favorites or the program search list. Essential Tools allows you to run easycheck, clean the instrument, or select Setup, where you can customize your list of favorites. Use Millipore to quickly access EMD Millipore websites for information.You can have up to seven assays at a time in your Favorites list. To add a favorite assay, select the assay from the Program Search list and click Add to Favorites. The new assay will be added to the bottom of the list. And if the list already consists of seven assays, the first assay in the list will be removed. To remove a favorite assay, click Setup under Essential Tools. Remove the check from the check box for the assay you wish to remove. Click DONE. 1-8 guava easycyte System User Guide You can create a Favorites list to quickly select an assay later. See FlowCellect Templates on page Essential Tools guava easycheck allows you to check the system s counting, fluorescence, and scatter performance prior to running samples. The guava easycheck Bead is a standard particle used with this tool. Cleaning allows you to run Guava Clean, an automated system cleaning cycle that cleans the fluid system. Setup allows administrators to: configure certain software features for specific users (see Appendix A: Administrator Features for more information) remove assays from your Favorites list enter assay registrations codes (see Unlocking guava InCyte on page 1-10) NOTE: The Setup button is available only when an administrator is logged onto the system. Millipore This section allows you to access commonly used EMD Millipore websites. You must have internet access.The system will notify you if you currently have the latest templates installed. 1 If updated templates are available, the following dialog appears. Click Yes to install the latest template package.
Click Export to download the templates to a USB drive. 2 Click Close when you receive confirmation that the latest templates were installed. If you chose to export the templates to a USB drive, select the drive from the dropdown menu and select Export. Exit Closes guavasoft software Unlocking guava InCyte guava InCyte requires an unlock key. If you get a message containing your computer code and prompting for an unlock key, EMD Millipore Technical Support and they will provide you with an unlock key for InCyte guava easycyte System User Guide Open a template before acquiring your samples in InCyte. The instrument settings, plots, gates, and markers for the assay are contained in the template. If you already acquired data using either InCyte or another guavasoft module, open the data file in the template and perform analysis using the Analysis Method provided. The assay template opens in InCyte. 2 Select the Analyse or Acquire button at the top of the control panel. Perform acquisition or analysis according to the instructions outlined in the kit user guide. If you are acquiring samples, the instrument settings should be close to what you need. Check the settings and fine tune, if necessary. If you are analyzing InCyte-acquired data, but you acquired the data without the use of a template, you will need to drag the template Analysis Method to the Analysed Group. Check the gates and markers and fine tune, if necessary. If you are analyzing a data file acquired using a program other than InCyte, open the data file. The assay template will contain the appropriate Analysis Method. You will need to create an Analysed Group and drag the data file and the Analysis guavasoft Software Overview 1-11 22 Method to the Analysed Group. Check the gates and markers and fine tune, if necessary.The guava InCyte module allows you to acquire and analyze up to ten fluorescence parameters in combination with forward scatter (FSC) and side scatter (SSC), as well as area, width, and time.
It provides automated compensation and an instant update feature, making it easier to perform complex analysis. Key Features A single Method (or analysis strategy) can be applied to multiple data sets in parallel, or multiple Methods can be applied to a single data set to obtain statistics to be displayed in the heat map. The process of creating equation-based gating schemes has been simplified through use of drag-and-drop regions; draggable features are used throughout InCyte. The HeatMap allows you to visually compare results using a plate map that displays varying shades of blue to represent relative statistical values. Results from up to six parameters or six data sets can be displayed simultaneously guava easycyte System User Guide 23 Additionally, all channels can be used with reagents that fluoresce in appropriate channels. Components of InCyte Files Data files acquired using InCyte contain raw FCS data, the analysis components also known as Methods (regions, gates, markers, statistics, etc), instrument settings, and compensation settings. The raw data combined with the Analysis Method make up the InCyte Analysed Group. At the start of acquisition, you have the option to select a Method, instrument settings, and compensation settings. These components can be retrieve from a saved InCyte FCS file. If you do not retrieve a Method, a default Method will be applied. Tool Bar The tool bar appearing on the far left side of the window provides additional features for acquisition and analysis. The features are also accessible from the Tools menu. Other than the Pie Legend, which is described on page 4-5, the tools are described in detail at guava InCyte Overview 1-13 24 the point in the workflow where they are used.Running other programs (even if you are not actively using them) during a run may interfere with acquisition or interrupt the run. The Acquisition screen appears when you enter the assay.
Use the Acquisition screen to acquire data from samples you run on the easycyte System. You can also perform data analysis from the Acquisition screen immediately following acquisition. Menu bar Switch between Acquire and Analyse control panels Button panel Tool bar Control panel - displays real-time acquisition parameters Plate map Plot tool bar 1-14 guava easycyte System User Guide 25 Acquisition Buttons Place the cursor over the icon to display text describing the button. Start New Session Adjust Settings Acquire Next Sample Save and Close Current Sample Next Step Retrieve Settings Save Settings Capillary Cleaning Tools Stop and Close Session File Abort The following table describes the acquisition buttons. Acquisition Button Start New Session File Adjust Settings Acquire Next Sample Save and Close Current Sample Next Step Retrieve Settings Description Allows you to select the worklist file, the Method, instrument settings, and compensation, prompts you to load the for acquisition or the adjust settings, if applicable. Allows you to adjust instrument settings using the appropriate sample. Begins data acquisition for the next sample. You can make changes to the sample information (except for Events to Acquire) after acquisition and before clicking Save and Close Current Sample. NOTE: When Autostart Acquisition is checked, data acquisition starts automatically each time a new tube is loaded. Therefore, you do not need to click Acquire Next Sample or Save and Close Current Sample. Saves the sample data. You can now acquire the next sample. Proceeds to the next step in the data acquisition process. The data already acquired is saved. Allows you to recall instrument and analysis settings from a settings file and download the settings to the easycyte instrument. If this button is disabled, your system administrator may have chosen to not allow the retrieval of Methods and instrument settings from individual Method and instrument settings files.