ChemoMetec SCC-100 - Manual (Page 24)

2 Introducing the NucleoCounter
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Figure 5 The SCC-Cassette. The fluorescent dye, propidium iodide,
is immobilized in the first part of the flow system.
2.2.1 Loading the cassette
The SCC-Cassette is loaded by gently pressing the white piston, which creates a partial
vacuum in the flow system. The tip of the SCC-Cassette must be immersed into a lyzate
mixture when pressing the piston, resulting in lyzate being loaded into the flow system.
Approximately 50µl is loaded into the flow system.
2.2.2 Fluorescent dye
The fluorescent dye, propidium iodide, is immobilized in the first part of the flow
system. As the lyzate is loaded into the SCC-Cassette, the immobilized propidium iodide
is dissolved immediately and mixed with the lyzate. Propidium iodide intercalates with
double stranded DNA and forms a fluorescent substance, absorbing green light and
emitting red light, which is utilized for detection of stained cells. Furthermore PI's
ability to fluoresce is enhanced significantly upon binding to DNA thereby substantially
enhancing selectivity.
During the analysis the stained mixture is transported through the flow system, where it
is mixed with the propidium iodide, towards the chamber where the actual cell counting
is performed. The precise volume analyzed, is determined individually for each cassette
during analysis. This volume of milk analysed is approximately 1 µl. After analysis the
SCC-Cassette is disposed of as biological waste.