Fig. 5. Increased intracellular Ca²⁺ activity and increased motility in cells treated with cGMP agonists or NO donors compared with cGMP inhibitors, see Movies 1-3 in supplementary material for more detailed information. The false color scale is the same as that described in Fig. 4. (A) Increased motility and Ca²⁺ levels after preincubation with a cGMP agonist. After a 30-minute incubation with 100 µM 8-pCPT-cGMP, many of the cells show elevated Ca²⁺ levels using Fluo3 (left). These cells are moving, as shown by subtraction of a 30-minute image from a image taken at 0 minutes (right). (B) Low motility and low Ca²⁺ levels after preincubation with a cGMP antagonist. After a 30-minute preincubation with 50 µM 8-pCPT-cGMP only one cell with high Ca²⁺ levels is seen (left). This cell is moving, as shown by subtraction of a 30-minute image from a image taken at 0 minutes (right). The moving cell is an atypical, fusiform cell and might represent a macrophage derivative rather than an osteoclast (see text). (C) Increased motility and Ca²⁺ after preincubation with the NO donor SNP. After a 30-minute exposure to 100 µM SNP, many cells show elevated Ca²⁺ levels by ratio imaging (left) and are moving, as shown by subtraction of a 32-minute image from a image taken at 0 minutes (right).