LSRII and FACSDiva
Startup
· Turn LSRII on
· Follow start-up procedure, which is listed on the side of the machine.
· Turn the computer on
· Open BD FACSDiva software
· Login
· Click “Keep CST settings”
Browser
· Click on “booklet” icon to make an experiment
· Open book before renaming, and name with last name and date of experiment
· Create specimen using “syringe” icon
· Name tube. Can add subsequent tube just before collection
Inspector
· Click on 5 log decades (if using primary cells)
Cytometer
· In the Browser, click on the arrow to the left of the tube in use; once clicked, should turn GREEN. This makes the green tabs in the cytometer window appear.
· Green Tabs:
o Status
§ If something is displayed in the status window, there is a problem with the machine – seek assistance from FACS tech!
o Parameters
§ Delete the fluorochromes that are not going to be used. Hold down shift key to select multiple fluorochromes; then press delete button
§ Make sure FSC and SSC are selected; Check off boxes for A, W, and H.
o Threshold
§ Number of events collected. If set FSC at 5000, anything less than 5000 will not be collected, while everything about 5000 will be.
o Laser
§ Check laser settings with yellow-post it note n side of computer – should be the same as what is displayed!
o Compensation
§ Come back to this tab after collecting single stains
o Ratio
§ ?
Global worksheet
· Draw plots with “dot plot” button.
· Change axes; examples:
o FSC (x) vs SSC (y)
o FITC (x) vs PE (y)
o FITC (x) vs APC (y)
o APC (x) vs PE (y)
Acquisition Dashboard
· Type in the number of events to collect, and the number of events to display.
· Do not change anything on the storage gate! You want to store each event because you can look at them later.
· Make sure dial is set at a true medium (turn dial all the way to the left, then make 5 full turns. This is medium). Run samples on medium to avoid clogs!
· Start collecting unstained control (acquire data). If you change the voltage of FSC or SSC (in parameters tab), you must start collecting again. Keep hitting reset button and changing voltage until you are satisfied with shape of profile.
· Now, press the record data button.
· Draw P1 gate (polygon gate) on FSC vs. SSC plot for unstained control.
· Right click on plots; select “show gate à P1”.
· Can change number of events to display (i.e. 100,000)
Compensation
· Draw quadrants (Q1,Q2, Q3, Q4) or 2 gates (P3,P4) to delineate the (+) and (-) populations.
o Ex. Sample is PE (+) but FITC (-)
· Right click on plot, and click show statistics hierarchy
· Adjust the (-) fluorochrome means so that they are the same, by clicking the compensation tab.
o Ex. If samples is PE (+) but FITC (-), click on tab that says:
-% fluorochrome
FITC PE
Such that you are adjusting the mean of FITC (neg. fluorochrome for this sample)
· Repeat for all conditions.
Signing off
· Export experiment and FCS files (be sure to rename fcs files as .fcs to distinguish from experiment files, as they have the same name)
· Burn data onto a CD or DVD
· Be sure to delete files from Browser in FACSDiva, once disc is burned.
· Logout of FACSDiva (so you don’t get charged for time that you aren’t there!
· Can analyze data on computers not hooked up to LSRII
Updated 1/5/10 by RK