26 BD High Throughput Sampler User’s Guide
Mixing
HTS mixing efficiency is impacted by the viscosity of the sample in the well.
Adjust the mixing volume, speed, and the number of mixes to obtain the most
homogeneous population for acquisition.
A greater number of mixes might allow you to decrease the mixing volume and
speed, but this could impact sample throughput. Increasing the mixing speed and
volume might improve throughput and mixing efficiency, but it could also
introduce air bubbles in the sample well. In addition, increased mixing speed
could compromise the separator bubble between the sample and sheath, resulting
in sporadic event rates and possibly higher carryover.
Use default values as a starting point (see Table 2-4 on page 40) with a mixing
volume of one-half the total sample volume. Note that samples are mixed less
effectively in flat-bottom wells than U- or V-bottom wells. When using flat-
bottom wells, you might want to mix the sample well before pipetting it into the
plate, and acquire the sample before it has a chance to settle.
Carryover
You can vary the amount of wash volume to minimize carryover. In general, a
greater wash volume results in less carryover, but the greater the volume, the
slower the system throughput.
The minimum volume is determined by the sample and mixing volume. To start
with, try a 400-µL wash for 10 µL of sample volume. As the sample and mixing
volumes decrease, the wash volume can also be reduced.
Throughput
The speed at which the HTS completes a plate depends on the acquisition time
(sample volume/sample rate), acquisition mode (high throughput or standard),
mixing volume and speed, number of mixes, and wash volume. To achieve
optimal throughput, BD recommends that you concentrate the sample, reduce
the sample volume (10 µL or less), increase the sample rate (1 µL/sec or greater),
minimize the number of mixes (2 or less), and decrease the wash volume (400 µL
or less).
NOTICE The BD FACSCanto II maximum event rate is 10,000 events/second.
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