8/30/2023 0 Comments Facs analysis![]() This is a very useful technique when searching for various discreet subpopulations. Notice that you may now switch between no gates ( None), the lymphs gate, and the NOTlymphs gate. How is it different? Right-click in the plot and choose the Gate option. Click OK to close the dialog boxes and look at your plot once more. Click the Gate Create/Edit button again, but this time choose the Not option as well as lymphs. Right-click on the plot again, and return to the Change Format window. How is your plot different?īy creating a gate using the lymphs region, the new dot plot is only showing those cells which fall within the lymphs gate. Select lymphs under regions and click OK to close both dialog boxes. A new dialog box opens (Create or Delete a Gate). In the Dot Plot Format window, choose Gate Create/Edit. Next, right-click in the center of the new plot and choose Change Format. Choose Norm.001 and set the Parameters to Fitc vs PE. Go back to the Display menu, and again choose New Dot Plot. At this point, your plot should resemble this: In the Create Region dialog box, name the region “ lymphs“. Use the mouse to draw a region around the lymphocyte population (draw a polygon by clicking the vertices click the starting point to close the region). Right-click in the center of the plot and choose Create a Region. The plot should be familiar to you from the previous lesson. Choose the following options:Ĭlick OK, and the software draws a bivariate dot plot. Locate and open the Norm.001 data file which you downloaded previously. Under the Edit menu is the Preferences option where you can customize the look and feel of the application.įrom the Display Menu choose New Dot Plot. If you are unsure or are curious about a feature, this is the first place you should look. Weasel has a very rich documentation under the Help menu. Take the time to familiarize yourself with the various options under each menu. Note the pull-down menus within the program. If you have purchased a license for the program, enter your registration code, otherwise click OK. Use the Weasel.jar file to start the application Linux/BSD: Functions identically to Windows version.When the instructions refer to a “right click”, simply press and hold the control or option keys while clicking to emulate a right click. Mac OSX: Weasel utilizes a two mouse-button scheme.Windows: Depending upon your version of windows, weasel should be started by the Weasel.bat (win9x/NT) or the Weasel.jar (XP/2000) file.If you need the files in another format please email me. To prevent corruption on download, the files have been archived in zip format. Be sure to contact M-Stores Customer Service prior to any purchase.įinally, download the data files to be used for this exercise. The University offers substantial discounts on many cytometry software packages. You may, of course, use any other cytometry analysis program which is already installed on your system. ( Note: If you are running on an HITS-administered workstation, you may need to contact the help desk in order to have the software installed). Java and Quicktime may also be required if not already present on your system. Please be sure to follow the installation instructions carefully. Please visit the Weasel download page and download the appropriate version for your computer. As it is java-based, it will run on most modern platforms. Weasel is a flow cytometry data analysis program available for download from the Walter and Eliza Hall Institute of Medical Research. As such, you will practice data analysis with a similar program known as Weasel. Wash once with PBS containing 0.1% saponin, remove supernatant and resuspend the cell pellet with 0.5 ml 2% formaldehyde solution.While the software package utilized within the flow core is CellQuest Pro, the software is dongle-protected and quite expensive.Wash cells 2-3x with FACS buffer and suspend in 200-300 microliters FACS buffer for analysis.You will need to determine the proper concentration for each antibody used. Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently.Suspend the pellet from the final wash in 50 microliters FACS buffer per each analysis on a single sample - up to three separate staining reactions can be set up from a single sample). Cells should be washed 2-3x with FACS buffer (PBS supplemented with either 1% BSA or 5% FBS and containing 0.05% NaN 3 ).(Alternatively, Becton-Dickinson sells a product called "FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells.) At MDS/RRDA, RBCs are lysed using either Gey's solution or a buffered ammonium chloride (ACK) solution. This can be accomplished by several means. Collect blood (75 microliters) into 1ml PBS containing 5 microM EDTA (10 microliters of 0.5 M stock) and mix immediately to prevent clotting.FACS Analysis Using Peripheral Blood Cells
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