Cytell Cell Imaging System Customer Instructions siglo Green BioApp

Cytell Cell Imaging System Customer Instructions siglo Green BioApp

2 Cytell Cell Imaging System siglo Green BioApp Customer Instructions

Cytell Cell Imaging System siglo Green BioApp Customer Instructions 3 siglo Green BioApp Customer Instructions Discusses important use and safety information Introduces the new siglo Green BioApp Describes how to install the siglo Green BioApp software Describes how to use the siglo Green BioApp on the Cytell Cell Imaging System Important user information WARNING Using controls, making adjustments, or performing procedures other than those specified in the Cytell Cell Imaging System s user documentation can result in hazardous exposure to high voltage, laser radiation, or moving parts. Exposure to these hazards can cause severe personal injury. Do not operate the Cytell Cell Imaging System in any other way than described in the user documentation. IMPORTANT! Prior to reading this document, you must read and understand the safety and operating information described in the Cytell Operating Instructions and the Cytell User s Reference Manual. Do not operate the Cytell system in any way other than described in the user documentation. If you do, you may be exposed to hazards that can lead to personal injury and you may cause damage to the equipment.

4 Cytell Cell Imaging System siglo Green BioApp Customer Instructions Intended Use Use of the Cytell Cell Imaging System assumes that you are familiar with the basics of fluorescence microscopy. In addition, an understanding of image processing basics will help you use the system to its full potential. To manage the computer system, familiarity with Windows-based personal computers is also helpful. About siglo Transfection Indicators Synthetic sirnas are the most commonly used RNAi tools for inducing endogenous gene silencing. The availability of large collections of pre-designed sirnas has facilitated high-throughput sirna library screening. This functional analysis approach has become a key discovery methodology in disease onset and progression, drug target discovery, host pathogen interactions, and pathway analysis. Determining the correct transfection conditions is the first step in designing a successful sirna screen. In these instructions, we describe how the use of Cytell Cell Imaging System and Dharmacon siglo Transfection Indicators can be used to find optimal transfection conditions. Dharmacon siglo Green and Red Transfection Indicators (siglo Green and siglo Red) are fluorescent oligonucleotide duplexes that localize to the nucleus thus permitting simple visual assessment of transfection efficiency. They are excellent for (1) use in experiments to determine optimal sirna transfection conditions (2) tracking individually transfected cells and (3) monitoring relative efficiency of delivery when co-transfected with sirna. They are not, however, intended for quantitative determination of sirna uptake. Installing the siglo Green BioApp Important! Cytell software version 3.2.6.12 or later must be installed on your system in order to properly install and use the siglo Green BioApp software. 1. Close the Cytell software. 2. Right-click on server icon in the taskbar and then click Exit to close the server. 3. Download the installation program for the new siglo Green BioApp software from the Cytell software download site: http://incelldownload.gehealthcare.com/bin/download_data/cytell/bioapps/sigl O_Green/

Cytell Cell Imaging System siglo Green BioApp Customer Instructions 5 4. Right-click the Cytell siglo Green.exe file on the desktop and select Run as administrator. 5. As the installation program runs, accept all of the default settings. 6. When the installation is complete, run the newly updated Cytell software. Once the software opens, you will see the new siglo Green icon on the home page. The software installation and update process is complete. Use the software as you normally would. Review the following section to learn how to create and run a new siglo Green protocol.

6 Cytell Cell Imaging System siglo Green BioApp Customer Instructions Creating and Running a New siglo Green Protocol 1. Select the siglo Green BioApp icon on the home page. 2. Select the New Data button to display the siglo Green Protocols page. 3. Select the preconfigured siglo Green Protocol icon and select Edit. 4. Select the Holder icon on the Plate Settings tab of the Edit Protocol page to display the list of sample holders.

Cytell Cell Imaging System siglo Green BioApp Customer Instructions 7 5. Select a sample holder from the list and place the holder on the stage. Select Done. 6. Select the well(s) to scan and, if desired, enter a Protocol Description. Select Next. 7. On the Sample Imaging tab, one acquisition field will be displayed in the sample imaging area within the well. Add fields and modify their positions as necessary using the sample imaging tools located along the left side of the sample imaging area.

8 Cytell Cell Imaging System siglo Green BioApp Customer Instructions 8. Snap images for each selected wavelength (All Nuclei and Transfection) by selecting the appropriate camera icon. The Focus and Exposure controls will appear and the images will be displayed in the sample imaging area. Use the Focus and Exposure sliders to optimize the focus and exposure of each image. Once you are satisfied with the focus and exposure settings, select Next.

Cytell Cell Imaging System siglo Green BioApp Customer Instructions 9 9. On the Sample Analysis tab, select the camera icons from left to right, starting with the Find All Nuclei camera icon first, and then moving to the Find Transfection icon. As each target is identified, the images will be displayed in the sample imaging area and the locations of nuclei and cells will be highlighted. Use the Nuc Area and Sensitivity sliders to correctly identify nuclei. Use the Show/Hide Masks icon to toggle the object masks on and off to assess the accuracy of identification and optimize the analysis as necessary.

10 Cytell Cell Imaging System siglo Green BioApp Customer Instructions To correctly identify Transfection positive cells, move the Threshold slider to gate on an appropriate intensity value. This setting can be fine-tuned following imaging after all the data has been collected. When finished with the Sample Analysis tab, select the Next button to save the protocol. 10. On the Run Protocol tab, confirm the well(s) to scan and then select Run.

Cytell Cell Imaging System siglo Green BioApp Customer Instructions 11 11. Choose a library location to save the data, enter a protocol run name, and select OK to run the protocol. The protocol will run and generate the data and reports. View them using the same methods used to view data and reports created by running other protocols. Please refer to the Application Note Determining transfection conditions using siglo Transfection Indicators and the Cytell Cell Imaging System for additional experimental details and results using Dharmacon siglo Transfection Indicators and Cytell Cell Imaging System found on the Cytell Resource Center. http://www.gelifesciences.com/gels/campaigns/cytell-cell-imaging-systemresource-center

GE and the GE monogram are trademarks of General Electric Company. Cytell, Dharmacon, and siglo are trademarks of General Electric Company or one of its subsidiaries. All other third party names and trademarks referred to in this document are the property of their respective owners. 2015 General Electric Company All rights reserved. First published July 2015. For local office contact information, visit: www.gelifesciences.com/contact GE Healthcare UK Limited Amersham Place, Little Chalfont Buckinghamshire, HP7 9NA UK www.gelifesciences.com License agreement Any use of this software is subject to GE Healthcare Standard Software End-User License Agreement for Life Sciences Software Products. A copy of this Standard Software End-User License is available on request. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available upon request. Contact your local GE Healthcare representative for the most current information. Notice to purchaser The Cytell Imaging System software is for research purposes only. It is not approved for diagnosis of disease in humans or animals. GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D79111 Freiburg, Germany GE Healthcare Bio-Sciences Corporation 800 Centennial Avenue, P.O. Box 1327 Piscataway, NJ 08855-1327, USA GE Healthcare Japan Corporation Sanken Bldg., 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan