Aria Training

Troubleshooting the Stream
Acquisition Troubleshooting
Fluidics Troubleshooting
Electronics Troubleshooting

Troubleshooting the Stream

Observation	Possible Causes	Recommended Solutions
Stream not in center of aspirator	Difference in keyed stream position between nozzles	If you have just changed the nozzle, use an Allen wrench to loosen the screws on either side of the sort block. Adjust the angle of the sort block until the stream flows into the center of the waste aspirator, and then tighten the screws. See Figure 1-11 on page 32.
	Nozzle inserted improperly	Turn off the stream. Remove the nozzle and ensure that the O-ring is in place. Reinsert the nozzle at a slight downward angle to prevent loss or movement of the O-ring. Make sure the nozzle is completely seated against the back wall of the cuvette flow cell.
	Clogged or damaged nozzle	Turn off the stream, remove the nozzle, and examine the nozzle tip under a microscope. If debris is visible, clean the nozzle. See Cleaning a Nozzle on page 188. If the nozzle appears damaged, replace it. See Changing the Nozzle on page 140.
No stream or dripping stream	Grounding plate inserted improperly	Make sure the grounding plate is inserted with the notched end toward you. See Cleaning the Nozzle and Grounding Plate on page 165.
	Clogged or damaged nozzle	Turn off the stream, remove the nozzle, and examine the nozzle ti under a microscope. If debris is visible, clean the nozzle. See Cleaning a Nozzle page 188. If the nozzle appears damaged replace it. See Changing the Nozzle on page 140.
Stream control disabled or no stream when Stream control clicked	Plenum not full	Wait for the plenum to fill.
	Air lock in filter	Prime the system with the corresponding fluid. If the control is still disabled, remove the filter, install bypass tubing, and repeat the priming procedure until you see fluid in the line. When fluid is running through the line, remove the bypass tubing, install the filter. and repeat the procedure.
No stream when Stream control clicked	Sheath container low or empty	Refill the sheath tank. See Refilling Containers on page 103. Note that when the empty tank warning message is not dismissed after 15 minutes, the stream shuts off automatically.
	Air in sheath line	Prime the sheath tank. See Prime After Tank Refill on page 172.
	Air in filter	Purge the filter. See Purging Filters on page 182.
	Dry filter	Install bypass tubing in place of the filter for the affected fluid, and run Prime After Tank Refill. Reinstall the filter and open the bleeder valve to purge the filter. See Purging Filters on page 182.
Fanning around center stream	Nozzle inserted improperly	Reinsert the nozzle. Push it gently all the way forward without rocking it from side to side.
Unstable stream	Debris in flow cell or nozzle	Remove the nozzle and run the stream with no nozzle in place for approximately 1 second. (Click the Stream control on, and then off.) Sonicate the nozzle and reinstall it.
Leaking or spraying around nozzle	Nozzle inserted improperly	Turn off the stream. Remove the nozzle and the grounding plate at thoroughly clean and dry the plat and the area around the plate. See Cleaning the Nozzle and Grounding Plate on page 165 for instruction:
	Extra a-ring blocking nozzle	Remove the nozzle and use a cotton swab to clear out the cuvette.
	Grounding plate inserted improperly	Make sure the grounding plate is inserted with the notched end toward you. See Cleaning the Nozzle and Grounding Plate on page 165.
Drop breakoff too long	Bubbles in flow cell	Open the flow cell access door and check for bubbles in the flow cell. they are visible, turn off the stream wait a few seconds, and turn on the stream again.
	Attenuation on	Turn off attenuation.
	Amplitude too low	Increase the amplitude until you c see drops. If you need a very high amplitude (>70 volts) to see drop you might have air bubbles in the flow cell.

Acquisition Troubleshooting

Observation	Possible Causes	Recommended Solutions
No events in plots after clicking Load or Acquire	Acquisition pointer not set to current Tube	Click to move the Acquisition pointer in front of the appropriate Tube.
	Laser shutter engaged	Make sure the flow cell access do is completely closed.
	Laser power off	Turn on the laser power.
	Viewing plots for a different Tube	Double-click the current Tube in Browser to display the plots for t] Tube.
	Uncolored events in plot	Format the plot to display all events. Assign a color to the population displayed in the plot. Verify the population drawing order.
	Current Instrument Configuration different from optical setup	Verify that the instrument optics setup matches the current Instrument Configuration. See Application Options on page 220.
	No sample in tube	Add sample to tube or install new sample tube.
	Sample not mixed properly	Increase the Sample Agitation rate. See Sample Agitation on page 81.
	Threshold not set to correct parameter (usually FSC)	Set the threshold to the correct parameter for your application.
	Multiple Threshold parameters not set correctly	Verify that the correct Boolean logic (And/Or) was used for the Threshold parameters.
	Threshold channel too low or too high	Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 124.
	Optical filter(s) not completely seated	Make sure the filters are pushed all the way in.
No fluorescent signal	Current Instrument Configuration different from optical setup	Verify that the instrument optics setup matches the current Instrument Configuration.
	Wrong filter installed or filter not completely seated	Make sure the appropriate filter is installed for each fluorochrome; se Application Options on page 220 f suggestions. Make sure the filters a pushed all the way in.
	Laser delay set incorrectly	Adjust the laser delay settings. See Instrument Quality Control on page 105.
Low Area signal	Area Scaling factor too low	Adjust Area Scaling for the corresponding laser. See Instrument Quality Control on page 105.
Unexpected events in plot	Incorrect logic in Population Hierarchy	Verify the gating strategy.
	Incorrect population(s) in plot	Right-click the plot and choose Show Populations. Verify that the appropriate populations are displayed.
	Incorrect drawing order	Verify that the required population is not hidden by another population Right-click the plot and choose Order Populations by Count.
Unexpectedly high event rate	Threshold channel too low	Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 124.
	Sample too concentrated	Dilute the sample.
	Event rate too high	Decrease the Flow Rate in the Acquisition Controls frame.
	Bubbles in flow cell	Turn off the stream, wait a few seconds, and turn on the stream again.
Unexpectedly low event rate	Sample not adequately mixed	Increase the Sample Agitation rate. See Sample Agitation on page 81.
	Threshold channel too high	Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 124.
	Sample too dilute	Concentrate the sample.
	Sample line clogged	Perform a sample line backflush. See Sample Line Backflush on page 170. If necessary, change the sample line.
	Sample line clogged or kinked	Backflush the sample line. See Sample Line Backflush on page 170. Look for visible kinks in the line. If kinks are noted, change the sample.
Distorted parameters or high CV s	Instrument settings adjusted incorrectly	Optimize the scatter parameters. Adjusting the Voltages and Threshold on page 124.
	Flow rate too high	Decrease the Flow Rate in the Acquisition Controls frame.
	Window Extension too low	Increase the Window Extension.
	Bubbles in flow cell	Turn off the stream, wait a few seconds, and turn on the stream again.
	Nozzle clogged or dirty	Clean the nozzle as described in Cleaning a Nozzle on page 188.
	Flow cell dirty	Clean the flow cell with a detergent such as Contrad. See Clean Flow Cell on page 171. Let the detergent sit 5 minutes before turning on the stream.
	Poor sample preparation	Repeat sample preparation.
	Area scaling factor too low	Verify area scaling. See Verifying Area Scaling and Laser Delay on page 112.
Excessive amount of debris	Threshold channel too low in plots	Increase the Threshold channel. Adjusting the Voltages and Threshold on page 124.
High electronic abort rate (>10% of system event rate)	Window Extension too high	Decrease the Window Extension.
	Threshold channel too low	Increase the threshold channel.
	Event rate too high	Decrease the Flow Rate in the Acquisition Controls frame.
	Sample aggregated	Filter the sample.
	Sample too concentrated	Dilute the sample.
Fewer events than expected in gated population	Window Extension set incorrectly	Adjust the Window Extension. Refer to the BD FACSDiVa Software User's Guide, if needed.
	Laser delay set incorrectly	Adjust the laser delay settings. See Instrument Quality Control on page 105.
	Plot zoomed	Unzoom the plot or make the gate bigger.
	Events left out of gate	When drawing a gate, make sure events on the axis are included.
Increasing threshold results in decreased Area signal	Window Extension too low	Slightly increase the Window Extension to maximize Area signal. NOTICE Increasing the Window Extension too much results in more electronic aborts

Fluidics Cart Troubleshooting

Observation	Possible Causes	Recommended Solutions
No fluid in line during system prime	Air lock in filter	Remove the filter for the corresponding fluid, install bypass tubing, and run Prime After Tank Refill. Repeat the priming procedure until you see fluid in the line. When fluid is running through the line, remove the bypass tubing, install the filter, and repeat the priming procedure one last time.
Long clean fails	Air lock in filter	See previous recommendations.
	Fluid line detached	Verify the fluid line connections 0] the fluidics cart and on the instrument. Push firmly on each Ii] to ensure it is connected.
Fluidics cart air flow <70 psi	Air leak	Contact your BD Biosciences service engineer.
Fluidics cart air flow >100 psi	Regulator not adjusted properly	Contact your BD Biosciences service engineer.
Fluid leak under fluidics cart or below side door	Condensation from pressure relief valve	This is a normal phenomenon that occurs when water is condensed from room air. Condensation is greater in humid environments. To avoid slipping, check and wipe up the water daily.

Electronics Troubleshooting

Observation	Possible Causes	Recommended Solutions
"Instrument Disconnected" in Instrument frame	Instrument power off	Turn on the instrument main power.
	Communication failure between workstation and instrument	Quit the software and then restart it. If restarting does not work, reset the instrument electronics: switch off the main power, wait 10 seconds until the system is fully depressurized, and then switch the power back on. Restart the computer and the instrument.
	Ethernet cable disconnected between workstation and instrument	Unplug and then plug in the cable connectors and make sure they are secure.
	IP address changed	Enter the correct IP address. Call BD Biosciences for assistance.
"Master DAQ Overflow"	Event rate too high in Instrument frame	Decrease the event rate or verify the threshold.
"Instrument not responding" in Status tab	Unknown	Perform the suggestions for a communication failure, above.