Bio-Rad S3™ Cell Sorter - Manual (Page 69)

Instruction Manual | 59
5.2 Compensation
5.2.2 Manual Compensation
It is advised to use the auto compensation feature of the software, however it is possible
to alter the compensation matrix manually (Figure 65). This process, although historically
used, is difficult to set correctly and the process of compensation should only be performed
using the wizard unless knowledgeable and statistically correct adjustments can be done
by manual compensation. Compensation controls for each fluorochrome you will be using in
your experiment. The process is most accurate if you have both positive and negative events
in each sample, and it is advisable to acquire at least 5,000 positive events. After the sample
is acquired, load each sample one by one into the software, setting the histogram axes to the
parameters you would like to adjust, and checking the Comp box on the histogram axes. By
applying quadrants it is possible to attempt to match the median of the negative population to
the positive population (Figure 64). When the value in the matrix is changed, the populations will
move. Alter until the median values are correct and matched.
Fig. 65. Compensation matrix.
Fig. 64. Density plot showing hyperlog, log, and compensation options.