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　　We have prepared the propreitary lysis utilizing NCP.
＜Before adding NCL＞
＜5 minutes after adding NCL＞


<Measurement of the Dissolution Effet of NanoCulture®Lysis Using the KIT>

Measurement of the spheroid cell number
5 minutes after adding NCL to the culture medium, spheroid dissolves.*
Measure the cell number following the kit protocol**
*Spheroid cultivated by other cultivation method than NCP may not dissolve.
*Correspond to Promega, CellTiterGro, Invitrogen DNA PicoGreen.

Analytical Curve When Adding NCL
The analytical curve created for the DNA-cell dilution series
using NCL on Invitrogen's Picogreen indicated linearity in a wide range.


The analytical curve created for the ATP-cell dilution series
using NCL on Promega's CellTiter Glo indicated linearity in a wide range.

HT29 was submitted to the spheroid cultivation procedure at various seeding densities.
On the 5th day of the cultivation, the spheroid was dissolved with NCL
and the cell number was measured for both systems.
The analytical curves were created from the cell dilution series
and the DNA/ATP amounts were converted into the cell number.

The CelltitterGlo (ATP) system's cell number was estimated to be lower
than that of the Picogreen (DNA) system.
Here the implication is that the intracellular ATP amount decreases
when employing spheroid cultivation.

The CelltitterGlo (ATP) system is suitable to relative measurement,
whereas the Picogreen (DNA) system can also used for measureing the absolute number.

Example of Using NCL

NCP teatment was applied to both monolayer-cultivated cells and spheroid-cultivated cells
and cell numbers were measured using NCL for CelltitterGlo.
When NCP treatment was started after enough spheroid was formed,
the reactivity of the agent between monolayer-cultivated cells and spheroid-cultivated cells.