ChemoMetec YC-100 - Manual (Page 24)

2 Introducing the NucleoCounter
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Figure Figure Figure Figure 5 55 5 The NucleoCassette. The fluorescent dye, propidium iodide, is immobilized in
the first part of the flow system.
2.2.1 Loading the cassette
The NucleoCassette is loaded by gently pressing the white piston, which creates a
partial vacuum in the flow system. The tip of the NucleoCassette 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 stabilized lyzate is loaded into the NucleoCassette, the immobilized
propidium iodide is dissolved immediately and mixed with the lyzate. Propidium iodide
intercalates with DNA and forms a fluorescent stain, 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 is approximately 1 µl, and thus the volume analyzed is 10-
20 times that of a conventional hemacytometer. After analysis the NucleoCassette is
disposed of as biological waste.
2.2.3 Sample volume
During production each cassette is marked with a dot code, which specify the precise
depth or thickness of the measurement chamber of the cassette. The dot code is read
and decoded in the instrument during analysis and the volume analysed is determined