2.4. Live imaging
2.5. Data analysis and mathematical modeling
Images at individual time points of all organoids were quantitatively assessed using the SoftWoRx (Applied Precision). For individual images, circular areas with LBH589 diameter of 30 μm were manually set inside (luminal space) and outside (culture medium) of the organoid, and the fluorescent signal for each compartment was expressed by average pixel values of the circular area. The outer intensities (culture medium) of the images acquired immediately before (0 μM) and after (1 μM) the Rh123 addition were applied for calculating the concentration of all other areas of a given experiment.
For mathematical modeling, we made an assumption that Rh123 concentration in the lumen is determined only by active inward transport and passive bidirectional diffusion, both of which take place at the inner apical membrane. When Rh123 concentration (1 μM) remains stable in the donor side due to the excessively large volume in space, the rate of active transport in the basal to apical direction, Vin (nmol/s), can be expressed as below:
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