Page 85 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

33342 (Invitrogen; 5 μg/ml final concentration) were added to the Opticell chamber. Microscopic examination was performed directly after the PI and Hoechst addition with a fluorescent microscope (Olympus) equipped with the same setup as applied for SPIO labeling measurements, only that a 5× LMPlanFl (NA 0.13) objective (Olympus) was used here. For each condition five different fields of view were acquired (~2900 cells) within the 6 mm circle around the center of the insonified area. Different filter sets (U-MWU2, 330 – 385/420 nm; U-MWIB2, 460 – 490/510 nm; U-MWG2, 510 – 550/570 nm, Olympus) were applied for detecting all cells (stained with Hoechst), viable cells (stained with calcein-AM), and dead cells (stained with PI) respectively. All images were automatically analyzed in ImageJ [53]. The Find Maxima function in ImageJ was used to define the exact number of cells. To find an appropriate tolerance for the Find Maxima function in every image, the number of local maxima was defined for tolerance parameters of 0 to 200 in steps of two. We analyzed the differences in number of maxima between the steps. When the difference became smaller than 20, this point was considered as the correct tolerance and the corresponding number of cells as correct number of cells. This approach was validated by selective manual counting of number of cells (n=10). The difference between manual and automatic counting was 2.1±0.4%. As shown in S1 Fig, the % of viable cells determined from the calcein-AM staining (live cells) was the same as the cell viability determined from the PI staining (dead cells). The cell viability data was therefore presented as the % of viable cells determined from the calcein- AM staining.
SPIO cell labeling using ultrasound
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