Bio-Rad S3™ Cell Sorter - Manual (Page 65)

Instruction Manual | 55
5.2 Compensation
5.1.8 Pausing and Resuming
Acquisition may be paused and resumed while in progress using buttons on the instrument
control panel. Pausing will stop the sample flow. During the pause, the time parameter within
the data file will continue, and will show a time gap when resumed.
If you wish to replace the sample tube during the pause:
1. Press the touch locking system screen above the loading stage until the padlock is
displayed as unlocked.
2. Move the loading stage into the wash position.
3. Replace the sample.
4. Move the loading stage into the run position.
To resume, click the Resume button on the instrument control panel.
IMPORTANT! If the loading stage is not placed in the wash position after a sample is
removed and before a new one is loaded, the system will not provide a pressure
boost. Without washing the sample line, contamination in the new sample may occur.
5.1.9 Stopping and Saving
When the desired data have been collected, acquisition can be stopped and the data saved.
The data are saved in FCS 3.1 format and will include the protocol used for acquisition. The
stop button will depressurize and unlock the sample. Before running another sample, place the
loading stage in the wash position to backflush and clean the sample line.
5.1.10 Backflushing and Washing
After the sample is unlocked, the loading stage can be moved to the wash position. The wash
process includes a backflush and an exterior wash of the sample line. This process is crucial to
reducing carryover and for overall cleanliness of the system. After washing, the loading stage
will automatically unlock, and the next sample may be placed into the run position.
If an additional wash is desired, you have two options:
n
Lower the loading stage for >2 sec then bring it back to the wash position. An additional
wash cycle will run after returning to the wash position
n
Press the touch locking system screen above the loading stage until the padlock displays as
locked. This will initiate an additional wash
5.2 Compensation
Compensation is the process by which spectral overlap (spillover) is removed from other
detectors. The inherent overlap of emission spectra from fluorescent labels and probes makes
compensation necessary. This is of particular importance when simultaneously measuring
fluorescence from several labeled antibodies or probes. During acquisition, compensation can
be adjusted to eliminate this spectral overlap between fluorophores (Figure 59).
The density plot shown displays an FL1 control data file (Figure 59). The FL1 positive population
is higher on the FL2 scale than the negative population, though it has no FL2 fluorescence.
When compensation has been applied, the positive FL1 population has the same median value
as the FL1 negative population in the FL2 parameter.