Fluidigm CyTOF XT - Mixing; Probe Line Stirring; Sample Loop Vibrations; Unclogging

Chapter 2: Mass Cytometry and CyTOF XT
CyTOF XT System Components Overview
CyTOF XT User Guide 27
Mixing
Mixing occurs as part of the pellet resuspension workflow, and prior to loading samples.
During mixing, the probe line and fill pump work together to act like a pipette, aspirating and
dispensing a volume between 25 and 250 µL multiple times within a tube. You can select a
mixing force value between 1 and 10 to define how fast this volume is ejected into the
sample tube, with 1 representing the lowest mixing force and 10 representing the strongest.
The default setting for this parameter is 10.
NOTE We recommend using a mixing force of 10 and optimizing it for fragile sample types.
Probe Line Stirring
During sample acquisition, the probe line stirs the sample to prevent cells from settling. You
can select a stirring speed value between 0 and 9 to define how fast the probe line spins,
where 0 turns off the stirring and 9 is the fastest speed. The default setting at which the
probe stirs the sample is 5. During acquisition, the probe line is washed both inside and
outside when a pre-wash cycle is selected.
Sample Loop Vibrations
The default sample loop vibrations setting helps mitigate cell settling within the sample loop.
We do not recommend changing this setting unless running fragile sample types.
Unclogging
The Autosampler Module has automated clog detection and removal workflows. There are
2 pressure sensors on the Autosampler, 1 on the push pump and 1 on the fill pump. These
sensors are used to monitor pressure. During sample injection, the software takes readings
from the push pump every 56 sec. If the average pressure for 5 consecutive readings
reaches a set threshold, the software reports that a clog is detected. Acquisition then
pauses, and an automatic unclogging routine starts.
The Autosampler begins the automated clog sensing workflow by switching from injection
mode to load mode, which enables the push pump to be used to flush the nebulizer while
the sample in the sample loop remains undisturbed. An initial attempt to remove the clog is
done by pulse aspirating and flushing at high flow for about 12 min. If the clog is not
removed, the software then runs a clog identification workflow, in which the 2 pressure
sensors are used to determine whether the clog is in the sample loop or downstream of the
sample loop (for example, in the nebulizer line or the nebulizer). The unclogging procedure
then focuses on the location of the clog. During the unclogging, the system increases the
flow rate level and the pulse rate from light to more vigorous.
If the clog is successfully removed using the automated workflow, acquisition resumes. If the
clog cannot be removed using the automated workflow, acquisition stops, and the sample is
returned to the sample tube (if Recover Sample is specified in the acquisition template). The
clog must then be removed manually. For more recommendations to reduce clogs and manual
intervention techniques during acquisition or after acquisition stops, click Help to see the
integrated help guide, also available as the CyTOF Software v8.0 Help for CyTOF XT (FLDM-00045).