Start Cellquest Pro. Choose connect to the Cytometer in the Acquire menu. Making a new acquisition document Alternatively screen files and data files that have been saved contain instrument settings and can be opened directly into the program by double clicking or opening from the file menu.
Create an acquisition dot plot with FSC and SSC as the x and yClick and drag the dot plot tool on the toolbar onto the document and adjust the size of the plot. Plots menu- format plot to modify the size of this plot will determine the size of the other plots.
Acquisition histogram plot for single fluorescent FL1, 2, or 3 Acquisition dot plot arrow clicked below Acquisition density plot for dual parameters e. FL1 vs. By clicking on the axis of the Plots and using the menu to the right you will be able to assign the parameters you would like to measure. Prime the SIP several times before running the sample by pressing the prime button.
On the left below the SIP is a switch that indicates either a tube manual or a plate autosampler. Make sure it is turned toward the tube setting. When not running the sample leave the instrument on standby.
For SSC and fluor. Adjust the voltage. Clarify your population on the screen. Adjust the voltages and amp. To copy settings from one well to another, click to select the well, then right-click it and choose Copy Cytometer Settings.
Select the well you wish to copy to, then right-click it and choose Paste Cytometer Settings. See Running Samples Automatically in Sequence in the following section and Running Wells Manually on page 47 for a description of each mode.
Data collection proceeds automatically according to sequence number. Select noncontiguous samples by holding down the Ctrl-key while clicking individual specimens, or select a range of samples using the mouse.
Wells are run in the following order: setup controls compensation controls specimen wells NOTICE When a sequence is in progress, the ability to select a well in the plate layout is disabled. The system flushes any remaining sample that was aspirated.
Once the sequence run is complete, a dialog will be displayed with the status of the run and whether any errors occurred. An alert will sound until the dialog is dismissed. You can acquire, record, and restart acquisition on a single selected well as long as a sequence is not currently running.
Stopping time in manual acquisition mode is calculated the same as in automatic sequence mode. The software will stop acquisition or recording of a well when any of the following stopping rules have been met: the specified number of events were collected the stopping time was reached the file exceeded memory specifications acquisition was stopped by clicking the Stop Acquiring button under Basic Controls Once acquisition of the well is complete, any remaining sample is flushed from the system.
The controls in the Acquisition Dashboard are briefly disabled while the loader is reset. Analysis View Components The Analysis view provides an interface for displaying keyword results in the plate layout reviewing and analyzing data collected from a plate starting a batch analysis For information on performing these functions, see Analysis View Functions on page Plate Layout The plate layout in the default Analysis view displays the acquisition status of each well.
See Table on page 36 for status indicators. If keywords were assigned to the plate, clicking on a keyword in the Keywords list toggles the plate layout to show the Keywords Analysis view. See Figure Each keyword is shown as a different color. If a keyword was assigned a range of values, a value is displayed in each well.
Different values of the same keyword are shown in various shades of the keyword color. If a well has no value assigned, the well is white. To revert back to the default Analysis view, click the keyword in the Keywords list. The Available Keywords list displays all keywords that were used in the experiment.
To assign new keyword, see Assigning Keywords on page To view keywords in the Analysis view, you must add the Keywords to the Selected Keywords list. For complete instructions, see Displaying Keywords on page To save a record of analysis, print this view after analysis is complete. The printout shows the plate layout, legend with keywords, and plate name and type. Keywords List The Keywords list contains the selected keywords for all samples and wells on the plate Figure A maximum of 15 keywords can be added to the list.
Add keywords to the list using the Add Keyword button. See Add Keyword Button for more information. The well is outlined in blue, indicating the well is selected for analysis. In the Browser, the current tube pointer appears to the left of the plate name. See Figure for an example. Figure Analysis indicators selected well in Analysis view current plate or tube pointer Shortcut Menu Right-click a selected well at the Analysis view to open the shortcut menu.
Analysis View Functions Perform the following functions in the Analysis view. Use the Keyword Analysis view to display keyword values for all wells that were assigned the selected keyword. The Keyword list displays all keywords that were used in the current experiment. When you choose a keyword, the plate layout shows keyword values and corresponding background colors.
Wells are colored according to keyword assignments. For example, Figure displays values for the keyword Concentration. In this example, four different concentrations were assigned to wells on the plate. Hydrodynamic focusing forces sample cells through the cuvette flow cell, where they are interrogated. The flow cell is in fixed alignment with the laser and gel-coupled to the collection optics.
This design ensures that the laser is precisely focused on the sample stream and the maximum amount of emitted light can be collected for added sensitivity in multicolor applications. Fixed alignment also minimizes startup time, improves experiment-to-experiment reproducibility, and enables automated daily quality control QC. The sheath container 8 L and waste container 10 L are outside the cytometer, positioned on the floor for easy access. Fluidics sensors maintain constant pressure, while a fluidics monitoring system warns when sheath fluid is low or empty, or when the waste container is full.
Note that all peaks are easily identified in various detectors from the blue A, B , red C, D , and violet E, F lasers. This affords researchers greater application flexibility, allowing them to easily move assays from one platform to another. The software reduces operator error, and ensures consistency of results by setting the signal time delay across the multiple laser beams and optimizing PMT voltages. Application-specific settings can also be specified, allowing for rapid setup and performance of routine experiments in a more consistent manner.
QC tracking capabilities in the software measure instrument settings and report on performance. Levey-Jennings plots help users understand instrument performance and identify maintenance issues. Acquisition and Analysis BD FACSDiva software enables researchers to preview and record data from multiple samples with an automated acquisition process.
Acquisition templates, user-defined experiment layouts, and simple compensation procedures are also managed by the software to further facilitate acquisition. For analysis, the software includes powerful features such as hierarchical snap-to gating, a variety of plot formats, and batch analysis.
Recorded data can be analyzed by creating plots, gates, population hierarchies, and statistics views on a BD FACSDiva global worksheet. Once the global worksheet is saved, it can be used to analyze multiple sample tubes from an experiment, thereby saving time.This position also ensures that the aspirator arm is able to detect an installed sample coupler. Innovative designs for both the excitation optics and collection optics in the BD LSRFortessa X system minimize light loss and optimize collection efficiency for increased sensitivity and resolution. If a keyword was assigned a range of values, a value is displayed in each well. The sheath container 8 L and waste container 10 L are outside the cytometer, and positioned on the floor for easier access. For analysis, the software includes powerful features such as hierarchical snap-to gating, a variety of plot formats, and batch analysis. Numerous other productivity benefits include user-definable batch analysis and automated features such as gate resizing, pausing between data files, exporting statistics, and printing before proceeding to next data file.
Application specific settings can also be set, allowing for rapid setup and performance of routine experiments in a more consistent manner. This allows researchers to identify cells, especially dim and rare cell populations, optimizing multicolor assays and panel design for superior results. The following message appears. Use the Log scale when the sample has a large dynamic range of fluorescence signal as it has 4 decades of range. Slide the front cover to the left; slide the side cover to the back. Quality control QC tracking capabilities in the software measure instrument settings and report on performance.
The Mixing Volume is the volume of sample aspirated and dispensed during mixing.
Standard throughput mode can be selected for acquisition of larger sample volumes. Copying and Pasting Cytometer Settings You can copy and paste cytometer settings from one specimen or well to another as long as the specimen or well that you are copying from has cytometer settings. Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease. Each keyword is shown as a different color. The Operator is responsible for Collection Optics Emitted light from the gel-coupled cuvette is delivered by fiber optics to the detector arrays.
This allows researchers to identify cells, especially dim and rare cell populations, optimizing multicolor assays and panel design for superior results. The flow cell is gel-coupled to the collection optics to maximize detector signals. Fast acquisition speed is achieved by synchronizing two high-precision pumps for sample mixing, sample injection, and probe washing. Draw region of interest around events of interest. You can also select throughput mode in the Plate Inspector. If you export a data file or experiment that contains incomplete data, the software cannot recognize that the file is incomplete.
Application-specific settings can also be specified, allowing for rapid setup and performance of routine experiments in a more consistent manner.
Reboot the computer. Change the directory to your lab folder. The flow cell is gel-coupled to the collection optics to maximize detector signals. To adjust the background fluorescence FL1, FL2, FL3 Using the control sample, place the population in the first log decade by adjusting the appropriate fluor. NOTICE The throughput mode applies to the entire plate, so all wells on the plate are acquired in the same mode, except for setup and compensation control wells, which are always run in standard mode. Superior Performance While improved laser and optical detection design features have been incorporated into the BD LSRFortessaTM X, the fluidics system retains the true fixed-alignment flow cell design.
Create an acquisition dot plot with FSC and SSC as the x and yClick and drag the dot plot tool on the toolbar onto the document and adjust the size of the plot. Optics System The optics system consists of laser excitation optics that illuminate cells in the sample, and collection optics that direct light scatter and fluorescence signals through spectral filters to detectors. For complete instructions, see Displaying Keywords on page In addition to laser wavelength, the special order program offers multiple laser power options and filter selections to better optimize assay needs.
For analysis, the software includes powerful features such as hierarchical snap-to gating, a variety of plot formats, and batch analysis. Turn gate on in plot source menu. Move the aspirator arm to the left. You can also select throughput mode in the Plate Inspector.
You can move wells within a specimen to change the acquisition order or move wells from one specimen to another. Notice that some steps apply only to a specific cytometer s. This automatically adds a new well to the existing specimen in the direction selected. Fluidics sensors maintain constant pressure, while a fluidics monitoring system warns when sheath fluid is low or empty, or when the waste container is full.
Setting Up for Plate-Based Acquisition This section describes how to set up the cytometer for plate-based acquisition.
Multiple Power Options Cuvette flow cell Innovative laser options include the choice of solid state lasers across the full spectrum. The specimen will be pasted, if there is room on the plate. For an example of setting up and performing batch analysis, see Performing Batch Analysis on page Do not allow the system to run dry, as this could damage the HTS pumps. Once the global worksheet is saved, it can be used to analyze multiple sample tubes from an experiment, thereby saving time. For SSC and fluor.
Posted by DavidMitchell Select Free Bonus: 4 04 The Computer Paper - Ontario Edition - Issuu Issuu is a digital publishing platform that makes it simple to publish magazines, catalogs, newspapers, books, and more online. Adjust the voltages and amp. This design efficiently exploits the principle that light reflection is more efficient than light transmission for fluorescence signal detection. Do not allow the system to run dry, as this could damage the HTS pumps.