A quick guide for the eclipse analyzer
Configuration:
1. Channels:
FSC and SSC channels
5 Fluorescent channels
EV channel
2. Lasers:
488nm and 640nm
3. Loader
An advance loader ready to take:
40 FACS/1.5ml/0.2ml eppendorf tubes
96/384 wells plate
It also contains 8 wells for sample
preparation, adding Ab or PI, etc….
Tanks:
The instrument contains PBS, enzyme cleaner and waste tanks. The PBS and waste tanks are connected
to larger containers located on the liquid cart below the instrument. There is no need to handle the
waste and PBS small tanks inside the eclipse, just to check that the tanks on the liquid cart are
full/empty, respectively, before running an experiment.
If one does handle the small tanks, there is a small arrow on each tank that should face a small arrow
inside the instrument for correct orientation.
One could add PBS to the PBS tank on the liquid cart even when an experiment is running on the
instrument, if the need arises.
There is also a liquid filter that may need degasing when starting up the instrument.
Operation:
1.
2.
3.
4.
5.
6.
Turn on the instrument via the on switch on the instrument right side.
Double click the EC800 V1.3.4 software on the desktop
Choose “acquisition” mode and press “OK”.
The current Database that saves all the date is called SQL. Do not change that.
Press “full startup”.
Build a new worklist
a. Choose plate/plate number/name
b. Mark the wells to read
c. Choose the protocol you want
d. Press “add”
One can repeat steps a-d several times thus creating a different protocol for every well/group of
wells in the loader.
After you press “Add” the different tubes appear on the sample list. One can add names to each
sample.
7. Press “save” and the instrument will automatically run the worklist.
After the run is finished the worklist is saved together with the results in the SQL database. That is
why you can’t run another worklist with the same name.
If you want to work with an existing worklist, you need to copy it and press “select”. The software
will automatically add “copy1”, “copy2” and so on, to the worklist name.
The display tab:
You can choose on the right side of the screen the number of windows you want to open
You can choose Density windows by ticking the density checkbox.
You can monitor the rate of events, total count and cell count/ul.
You can set the “Stop criteria” using gated events, run vol., time and/or another parameter you choose.
One usually positions the EV gain on 2
The PMT settings on 5 or 6
The laser tab for the lasers you need
Press for compensation. A table is open with each channel vs. all the others. Press “Auto’ and when
you close the window he will give you all the compensation values.
The parameters tab
One can choose for each parameter if to use linear or logarithmic scales.
FL1 (logarithmic scale)
FL1-Lin (linear scale)
FL1-Peak Lin (height)
FL1 FC Lin/log (Fluorescence divided by cell volume to neutralize differences emanating from different
cell sizes. Good for internal markers and gives a better, narrower fluorescent peak)
FL1 FSN Lin/Log (Fluorescence divided by cell surface. Good for membrane markers)
! Each change in the display tab, the stop gate or the PMTs is saved on the protocol. That is why before
you run you will be asked if you want to save changes.
The option tab
The option tab is important when running many samples.
On can define what is expected in each sample thus setting the conditions that cause the instrument to
rerun a sample. For example, if we expect 200event/sec and it fails the instrument can flush/clean
automatically and try to rerun the sample
At the end of the run you can also get a report if there were problematic samples. In the display tab
there is a window that shows progress of the run and marks wells by different colors:
Green – ok
Red – unable to read
Orange- failed but was able to fix and read again
Black – currently reading the well
Analysis mode:
To switch to analysis mode one can press an icon at the right top side of the software screen
The software opens in analysis mode
One can open a worklist/run (one well)/multi run (several wells)
Plate monitor tab:
One can chose the statistic/region/parameter and observe the results as data or as a color map
Run tab:
Shows the plots of each well. If you change the PMT or the gate you can choose to save and will update
all the statistical data.
Statistics tab:
You can define the statistical parameters you want (for each fluorescent parameter) and export it to
excel.
Report tab
As in the run tab but for several wells on the same page
One can add statistics to the plots