Intensity
l
see Fluorescence imagery appears too dim
l
see Fluorescence is too bright, images have a contrasting color or appear flat
l
see One channel saturates while the others do not
l
see Scatter is too dim or bright or changes over time
l
see Large variation in brightfield intensity levels
l
see Brightfield intensity level sets incorrectly
Symptom
Possible Causes
Recommended Solutions
Unstable fluidics
(Air or clog in
system)
Air bubbles in the
sample
Make sure a sufficient sample volume is used, 50 μl is recommended. To
clear the air bubble: Run the purge bubbles script.
Detergents and foaming agents (such as FBS) can cause bubbles to form in
the lines. If these buffers are causing air in the system remove them from the
sample and re-suspend in dPBS. Run the purge bubbles script.
Air bubbles in fluid
lines
Run the sterilize script, followed by the startup script. Load calibration beads
and verify the system runs normally.
Clog in fluid lines
Filter the sample with a 70 μm nylon cell strainer. Run the sterilize script, fol-
lowed by the startup script. Load calibration beads and verify the system runs
normally.
Sample is too con-
centrated
Clumpy and viscous samples cause cavitation in the fluidic lines and create
bubbles. Dilute the sample to 1x10^7 cells/mL and strain the cells through a
70 μm nylon mesh. Run the purge bubbles script.
Inappropriate sheath
solution
Verify the sheath is dPBS. De-gas the sheath as appropriate. Third party
sheath buffers cannot be used on the FlowSight System.
Fluid lines are leaking
With the system powered down look for leaking sheath. Verify the fluid lines
mount snuggly into position. Call Luminex Technical Support.
Fluidics respond
sluggishly
Air buffer in the sheath
syringe is not correct
The sheath syringe should contain 2-4 mL of air to buffer the movement of
the pump’s microstepper motor. If too little air is present run the “start-up”
script.
Fluid lines are leaking
With the system powered down look for leaking sheath. Verify the fluid lines
mount snuggly into position. Call Luminex Technical Support.
Event rate slows
over time
Cells have settled in
the lines
Cells settle in the lines after 45-60 minutes of running, resulting in a drop in
cell event rate. Stop and save the acquisition. Return the remaining sample,
restore the sample volume to 30 μl and re-load the sample to continue
acquisition. Data can then be appended together in IDEAS
®
Software.
There is a clog or air
bubble in the system
Run the purge bubbles script from the instrument drop-down menu.
information, see Unstable fluidics (Air or clog in system)
Sample syringe is
empty
Load a fresh sample
Sheath syringe is
empty
Load sheath, then go to the instrument drop-down and run prime.
Fluid lines are leaking
With the system powered down look for leaking sheath. Verify the fluid lines
mount snuggly into position. Call Luminex Technical Support.
For Research Use Only. Not for use in diagnostic procedures.
43
Amnis
®
FlowSight
®
Imaging Flow Cytometer User Manual
