Flow+Cytometry

Fluor configurations


 * Flow Cytometry Protocol**
 * Note:** One carousel has 32 tube positions.

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 * Write your name on the calendar next to the computer to show that you have used the instrument. It is best to sign up a day or two before so that people can plan their experiments
 * Log in to the computer if necessary.
 * username: administrator
 * password: "password" or just nothing
 * Select software – CXP cytometer. The software has 3 widows (main window, resource explorer, and acquisition manager)

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 * The status of the instrument is at the bottom of the main window.
 * Log in to the software with your log in information and password.
 * Login Info:
 * username: CIM user
 * password: vanessa
 * If there is an error message in the bottom right corner of the main window such as "Sheath Level Error" take care of this.
 * Start up takes about 8 min
 * Make sure the flow cytometer has enough reagents
 * Click the sleep mode button (“zzz”) to open the hood of the instrument. The hood opens just by lifting the front panel of the instrument up.
 * Check the levels of the cleansing fluid and sheath fluid. If they are very low, then add more (don't add above black mark). The cleansing fluid and sheath fluid are the only two accessible parts of the cytometer.
 * The cleansing fluid is blue and found next to the instrument on the counter or in a drawer.
 * Sheath fluid is clear and is in a box near the cytometer as well.
 * Close hood when finished. The door latch may get stuck. Pull on middle arm knob to release.
 * To continue, exit sleep mode (unclick the “zzz” button?)
 * If necessary, empty waste container on the floor near the instrument by pouring the waste down the drain and replacing the container.
 * Cleanse the instrument
 * Load the carousel with 1 tube containing 10% bleach and water, and three tubes containing H20 in slots 1-4. There should be at least more than 1 mL of these solutions.
 * Clear the working list if necessary (one of the buttons in the main window)
 * [[image:clear_working_list_button.png]]
 * Once the cytometer shows a status of "Awaiting Sample", select panels (in the resource explorer) -> Common -> Cleanse. Click and drage the cleanse panel to the bottom right acquisition manager window. Enter the appropriate carousel number. Hit the big play button in the main window to start the cleanse
 * The cleanse takes about 8 min. The progress of the cleanse may be monitored from the status at the bottom of the main window as well as the position of the arrow in the worklist of the acquisition manager.
 * After the cleanse has finished, clear the working list.
 * Insert about 10 drops of “flow check” solution into a cytometer tube (flow check solution is located in refrigerator of tissue culture room right behind the door).
 * Go to protocols tab in the resource explorer and then common acquisition protocols (in the resource explorer) -> select QC 1L flow check pro (this protocol checks for FITC) and drag this down to the working list in the acquisition manager
 * Place flow check tube in a slot in the carousel
 * Make sure to set the carousel # and location in the software.
 * Hit the big play button in the main window to start the protocol
 * Note about sheath pressure error: if this error occurs as displayed on the bottomo right of the screen, make sure the caps of the sheath fluid and cleanse fluid are on proper and tight (but not too tight), and that the drawer for these reagents is closed all the way so that it clicks.
 * After the calibration is finished clear the working list again by clicking the green button with the little list on it in the main window.
 * Setup the software so that files of samples can be named.
 * File -> Workspace Preferences -> LMD filename. You may want to check Sample ID 1, 2, 3, or 4 fields to be a part of the filename. These fields can be given a name for each sample. You may want to unselect the "run number" and "tag number" if you don't want these to be a part of the filename.
 * Create a new protocol if desired.
 * Select file -> new protocol
 * Select the gear icon for the cytometer control window.
 * clear working list button: "F:\kurt\storage\CIM Research Folder\DR\2013\6-21-13\wiki_download\clear_working_list_button.png"
 * Don't change anything under auxiliary
 * forward scatter (FS) and side scatter (SS) should be set to linear for eukaryotic cells. All of the other parameters should be set to log. Not that forward scatter correlates with approximate cell size and side scatter correlates with the cell complexity or granularity
 * close the parameter box
 * Set the maximum total number of events (often about 100,000)
 * Set the time maximum (often about 300s or 5 min)
 * Set the voltage which changes the spread of the cells in the SS vs. FS dot plot. The voltage can be changed under the "detectors" option with arrow bar at right. You may want to adjust the voltage so that cell populations are not on the very edges of the SS vs. FS plot and as much as the population can be resolved as possible. You may want to play with "quick set" option while acquiring unstained/untreated cells.
 * it may be a good idea to keep the cytometer control window open so that settings can be adjusted later if desired
 * The flow rate speed can be adjusted in the main window from Low, Medium, and High. The lowest rate will result in the highest resolution and give you the highest probability of seeing one cell at a time (see figure below)
 * [[image:flow_rate_figure.png width="339" height="239"]]
 * Save the protocol. This protocol can be found later under Main Drive/CXP/users/(some type of folder called acquisition or something)/(the name of the protocol)
 * Load samples into carousel (may want to switch carousels from the one used to calibrate)
 * Each sample should have at least 500 uL (1 mL might be better)
 * Create new panel (if the panel you want doesn't already exist; it probably doesn't)
 * Clear working list
 * Click file -> new panel. Enter panel name. A new panel must be created every time we do a new run.
 * Enter the number of samples you have
 * Choose protocol you want to use. Unfortunately, you have to keep choosing the protocol you want to use for every sample in your run (kind of irritating)
 * Save panel.
 * Create a worklist
 * If you just barely created a new panel, you probably don't need to worry about creating a worklist since your newly created panel will likely automatically appear as a new worklist ready to run.
 * Choose the panel you want to use from common panels or your username and drag it down to the acquisition manager in the lower right to create a new worklist.
 * Enter carousel # and location in the worklist, as well as any other information setup to be added to the filename.
 * Click the play button to gather data for samples (each sample may take 5 min if that's what was set for the duration in the cytometer control window)
 * View data for samples
 * The data for the samples can be viewed as they are running.
 * A gate can be created around the cells of interest in the SS vs FS dot plot and these cells can be further analyzed. Gates are created by choosing one of the gate options at the top of the main window.
 * New plots can be made by choosing one of the plot options in the main window (color dot plot, density plot, and a histogram). Histograms are suitable for situations with only one dye.
 * Different fluors for the plot can be chosen (FL1, FL2, etc.). FL1 corresponds to the FITC dye, FL2 corresponds to PE, FL3 corresponds to PI. FL4 corresponds to APC. The list of dyes is taped to the flow cytometer.
 * You can choose which gate to use to get the data for the plot. The gate can be changed later by right clicking on the plot and selecting format plot and changing the gate options.
 * Obtaining data for samples
 * Samples are saved as .fcs (flow cytometry standard) or .lmd. The files are saved in the lmd format by default
 * Data files can be obtained from (Main Hard Drive)/CXP/Users/(Username)/LMD
 * The computer does have a USB drive in the back
 * Shut down
 * Clean the instrument
 * Clear the worklist by hitting the new worklist button
 * Select Panels -> Common -> Cleanse
 * Make sure there are 4 tubes in the carousel in positions 1-4 (10% bleach tube, and 3 water tubes). More than 3 mL is a good amount to have
 * May want to retrieve data files during the cleanse cycle.
 * Close the software
 * Hit the FC off logo on the desktop. You should hear the instrument shutting down.
 * There is no need to log off the computer

Original Word Document with these Notes