ViCell Instructions
Contents
1. below pm image Picks out objects from background Brightness 75 90 85 Increase includes more objects Decrease includes less objects Picks out objects from background Sharpness 60 200 100 Decrease to include more objects Increase to include less objects Spot Determines cell viability p 75 Increase value more cells counted as dead Brightness Decrease value more cells counted as live Ratio of the bright spot to total area Spot Area 1 20 5 Determines cell viability Increase value more cells counted as dead Decrease value more cells counted as live os Excludes dead cells so they are considered debris Minimum Circularity 0 0 5 0 0 excludes no dead cells 1 excludes all dead cells Ability of software to pick individual cells froma None low Decluster medium cluster of multiple cells med high None counts a cluster as 1 cell2. in the user manual Running a Sample All reagents including the trypan blue are internal to the instrument so all you need is your cell solution to get started Sample cups are located on the shelf above the instrument Cell samples can be suspended in any of the following solutions PBS isoton liquid used with old cell counter or media even if it contains phenol red See system specs to ensure you will be within the necessary volume and cell concentration ranges Log in sample LY ition tachor Samola lD if Save images T Frm results Export to Excel fiie _ Acd io mulirun Excel f le Sample Prep a Prepare samples in the TC hood and bring to bench in ependorf or conical tubes b Make the necessary dilutions to keep your cell concentration within the correct range At the lab bench add 600 uL of solution to sample cup Exact volume does NOT matter as the instrument sucks up entire solution and only uses 500 uL Place sample cup in next available carousel position Log in sample on the computer by clicking on the Log in sample button log in window shown below a Select cup position on carousel b Enter your Sample ID the software is smart and will increment for you with multiple samples c Choose a Cell type first time choose default Select a dilution factor e Click OK or Next sample to enter additional sample data once you start the queue you can continue to enter samples so it saves time to start the
3. Vi Cell XR Cell Viability Analyzer User Instructions Written by CMCresson 15 Feb 06 The Vi Cell XR Cell Viability Analyzer is a video imaging system used to analyze yeast insect and mammalian cells It automates the trypan blue exclusion protocol in which dead cells take up the dye while live cells do not and provides data on percent viability and cell counts Essentially the Vi Cell system is an automated hemocytometer unlike the multisizer which was simply a particle counter The Vi Cell takes up the sample and delivers it to a flow cell and camera for imaging where differences in the grey scale between live and dead cells is determined by the software For every sample you run the instrument requires 500 uL of sample volume which is mixed 1 1 with trypan blue and takes 50 images to determine the cell concentration and viability All of this data is output on the computer screen and can also be sent to a printer or excel spreadsheet for further analysis System Specs Cell Diameter Range 3 to 70 um Cell Concentration Range 5 x 10 to 1 x 10 cells mL Viability Range 0 100 Counting Accuracy 6 Sample Volume 0 5 mL to 2 0 mL Acquisition Time 2 minutes Getting Started with the Vi Cell Everyone who will be using the Vi Cell system must be trained by the system administrator In addition everyone will be given a user account and password to log on to the instrument There will be a main system log in which user act
4. a from each parameter such as size distribution viability per image etc 9 To add additional samples to the queue click on Log in Sample and repeat the process in Step 4 above You can only add 9 samples to the queue at any given time But you can continually add samples as carousel positions become available 10 When the sample run is complete the instrument will clean itself Note You cannot review data images until this process is complete and there are no more samples to be counted 11 The carousel will then rotate to check for additional samples At this time empty sample cups will be knocked into an internal waster container 12 Read general system information below to ensure that you follow proper waste guidelines Analyzing the Sample Run When the cell analysis is complete you can flip through the images to check the accuracy of the instrument in identifying live dead cells You will also want to look at the size distribution graph to ensure that the correct minimum size value was used If you would like to change any of the cell type parameters choose instrument from the top tool bar and scroll down to click on reanalyze At this point the default cell type window will open up and you can adjust the necessary parameters and click ok The cell images will then be analyzed using the new parameter values and the data will be displayed on the right side of the screen Reanalyzing a sample does not overwrite the original data
5. and the new data is not automatically saved So you need to choose save run from the main menu in the top tool bar to save data before closing If you would like to reanalyze a previous run then you can reload the data by clicking open run from the main menu Then select the text file from your data folder and the corresponding images will be automatically loaded as well Saving Data In the user preferences everyone will have their own directory in which data is automatically saved All image files appear as file folders containing tiff files of the individual images and the data is saved as a text file If selected during the sample log in process your data will also be output to an excel spreadsheet There is an example excel sheet with explanations in the Vi Cell user manual for your reference It is recommend that all images are saved during the setup period so that data can easily be reanalyzed to determine the best cell type parameters However as the hard drive will quickly fill up you will need to alter your preferences at a later date NOT to automatically save the images Please be respectful of how much space you taking up on the computer and remove any unnecessary files The computer is not currently connected to the internet so data will have to be transferred by floppy disk CD or USB memory stick at this time General System Information It is recommended that the Vi Cell instrument remains ON at all times Do NOT turn off the ins
6. ivity will be recorded So if you allow someone else to use your account then you will be the responsibility party in the use log if something happens to the instrument UROPs will not be given user accounts and will be the responsibility of their lab advisor The Vi Cell system default cell type may work for some cell lines in the lab and it is really the best place to start However since trypan blue uptake cell size and appearance are unique to each cell type you should run some tests to determine the best parameters with your particular cell line For cell lines that are used by many lab members and that need changes to the default parameters we will create a cell type that will be specific for those cells The cell types will appear in every user s menu The software contains a tutorial for creating your cell lines so this may be a helpful feature for more difficult cell lines In addition you can email cell images to Beckman Coulter and they can assist you in determining the best parameters for your particular cell line It is also important to be aware of the amount of time your samples will be sitting at room temperature waiting on the carousel Each sample takes 2 minutes to run and the carousel holds 12 samples You may not want to prepare more than 12 samples to run at any give time as the waiting could affect viability counts However as sensitivity to temperature and pH will vary for each cell line you may want to run controls bef
7. ore making a large number of samples The Vi Cell user manual will be located on the shelf above the instrument at all times Please consult the manual or the software help function if you have any questions If these sources do not provide the info you need then please contact Catherine cresson mit edu or 8 9488 This is a very expensive instrument and it will be heavily used by many different lab members so please take good care of the Vi Cell and follow all of the operating procedures The Software Click on the Vi Cell icon on the desktop to begin the software You will then need to login to the program with your user ID and password provided by the system administrator Use the Navigation Bar on the left side to determine what is displayed in the main window BE Camera Image allows for the viewing of sample images during and after a run Pee Image Autosampler Queue shows the completed samples along with the list of samples in the queue this is where you can modify remove samples that are Aulosampler already in the queue Ld Le Lae Cell Types displays windows with the information for each specific cell b type this is where you create or remove new cell types Call Typa Bioprocess the last icon represents a bioprocess there can be many of these icons that each represents an ongoing experiment in which data is collected over days or months for more details of setting up bioprocesses BO S0vicon see pg 49
8. pe and you will be given the option to add modify or delete a cell type 2 choose File from toolbar then click on Image Reanalyze and you will be given the option to add modify or delete a cell type 3 choose Instrument Reanalyze from toolbar then you will be given the option to add modify or delete a cell type Sample Prep Options Parameter Range Default Details See of times sample is mixed in Aspiration i 1to7 3 syringe set according to cell Cycles nee sensitivity amp clumpiness Trypan Blue of times sample and trypan blue bee 1to7 3 f Mixing are mixed in syringe Image Analysis Parameters The ranges given below are the normal recommended ranges for each parameter and default value refers to the parameter value in the Default Cell Type The default values are a good starting place and you should tweak individual parameters to determine their effects on the output values For the cell brightness parameter you will need to observe the image in binary mode to see the difference between parameter values Right click on the image then turn off annotation and then choose Binary Mode Return to normal view for all other parameters CMCresson 15 March 06 Parameter Range Default Details Images 1 100 50 Increase value for very low concentration samples If a particular cell is being problematic place i 10 40 a Size 3 70 um cursor over cell and size is displayed
9. queue first From the navigation menu choose Autosampler queue to see your samples in the queue From this screen you can also edit remove samples in the queue while a run is in progress Click on Start queue to begin sample analysis Once the run begins your sample will disappear from the queue and you will only see it on the main screen The bottom of the screen will tell you exactly what the instrument is doing i e mixing trypan blue loading flow cell etc and right side displays the run data i e image viable cell count Indicates which position in the carousel you are putting your sample in NOTE the current three positions at the back of the carousel do NOT appear in the menu Fos tion Each user will be able to set their preferences for the default setting Also we will create new cell types for common lab cell lines Date eeu The dilution factor will automatically be Time 3524011 AM added into the result calculation by the instrument Choices are 1 0 to 20 0 Comme rt Erowse Hest sample X Cancel 7 Click on Camera view in the navigation bar to see images as they are collected Cells outlined in green are counted as live and cell outlined in red are counted as dead 8 On the right side of the screen data is displayed for each image as it is collected and the overall average is updated as the run progresses There is also a graph that displays detailed dat
10. times When the reagents do run out once again the software will give you an alert Do NOT attempt to change the reagents on your own This is the most common problem that occurs with the instrument so you will need to locate Catherine to change the reagents for you If Catherine is not around then please see Christina HD or Lisa to change the reagents The instrument will be decontaminated once a month by a 50 bleach cleaning and the person in charge of the Vi Cell will take care of this process Vi CellXR Cell Image Analysis Scheme Decluster Debris nt BS sae Panel Viable nee Min Dead Circularity Cell ial CMCresson 15 March 06 Creating and Modifying Vi Cell Cell Types When you begin using the instrument it is best to select the Default Cell Type to analyze your sample Then you can adjust these parameters to fit the specific cell line you are measuring and create a unique cell type These cell types will show up in everyone s user account and there should be only one cell type for each cell line that we use in the lab Please talk with other lab members to set the parameters for a given cell line so that everyone can use the same cell type for a given cell line For additional details please see the Vi Cell User Manual or the cell type power point tutorial which is saved on the desktop of the Vi Cell computer with the extension pps To access the cell types 1 choose File from toolbar then click on Cell Ty
11. trument At the end of a run the sample cup is dumped into a bin inside the machine The software will alert you when the bin has become full To empty the bin open the side door remove the metal tray and dispose of the cups in a biohazard waste container Also all of the liquid waste is collected by the instrument When the waste container inside the instrument is full the software will alert you First add 50 bleach to the waste and then you should dispose of the waste in the proper hazardous waste container in the hood If there is no waste container please fill out a new waste disposal card by following the example that is taped inside the door of the Vi Cell The waste percentages listed on the card should be the same for all samples 0 2 trypan blue 1 sodium hypochlorite 15 isopropanol Jf you are suspending your cells in a solution that is also hazardous you need to write that information on the waste card as well every time you use the cell counter As for the instrument reagents these will be ordered from Beckman Coulter by the lab manager and stocked near the instrument Only the Beckman Coulter reagents are compatible with this instrument Do NOT use any other brand of trypan blue If we are running low on reagents you will need to place them on the order board in the lab The instrument contains a reagent fuel tank that displays the relative amount of reagents in the machine and also the remaining number of runs at all
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