## 4 Discussion
Future studies of additional cytokines and transcriptional signaling pathways such as NF-kappaB will reveal if these pathways are affected. No distinct dose dependence of secreted TNF-\(\upalpha\) and IL-6 as a function of particle association was observed for either of the particles. In addition, SA-MIPs treatment did not cause an enhanced cytokine secretion compared to the reference particles, suggesting a limited NF-kappaB mediated inflammatory responsewas induced for the SA-MIPs. These results imply that the SA-MIPs possess low-level inflammatory properties that are of the same magnitude as PLGA particles, commonly used as an _in vivo_ reference. Dying cells induce an inflammatory response due to the release of damage-associated molecular patterns (DAMPs) and thereby engagement with pathogen recognition receptors (PRRs) on viable cells [44; 45; 46]. A reduced number of cells was observed upon the counting of the cells after culturing in the presence of SA-MIPs (Figures 5a and 6) indicating a possible cytotoxic effect by the SA-MIPs. However, a reduction in the cell number was also observed upon culturing with the reference particles Alhydrogel and PLGA (Figure 5a). PLGA particles are principally regarded as low-toxicity particles used in applications _in vivo_[47; 48]. It was only in the presence of the highest concentration of Alhydrogel that a tendency of an increased release of LDH into the culture medium was noted (Figure 5b), indicating cell death. Release of LDH into the culture medium is a commonly used method to determine cell lysis and cell death [49; 50] and the absence or low amount of released LDH from the cells undoubtedly confirms that the reduced number of cells upon culture with the particles, especially the SA-MIPs, was not due to particles cytotoxicity. Most of our current knowledge of how cells react to different kinds of perturbation is based on analysis of the response of an entire cell population. Microscopy techniques such as confocal microscopy, immunofluorescence microscopy, phase contrast microscopy and DHC are used to follow the behavior of living individual cells through time-lapse imaging. To scrutinize the effect of SA-MIPs on individual cells, DHC was used because of several advantages: low phototoxicity, label-free and quantitative data can be obtained on a single-cell level as well as on whole-cell populations [51]. Cell proliferation was studied for 48 h and showed a large reduction in the total number of RAW 264.6 cells in the presence of SA-MIPs, compared to cells grown in cell culture medium alone (Figure 6c). These results are well in agreement with the results obtained upon incubation of THP-1 cells with the examined particles and showed the effect of the particles on the whole population of the cells. DHC also enables longitudinal tracking of individual cells to observe cell-to-cell variability within a population. This has previously been used to show that the JIMT-1 breast cancer cell line contains a subpopulation of cells with a decreased response to salmonycin compared to the rest of the population [25]. Longitudinal tracking of single RAW 264.7 cells incubated with SA-MIPs showed that most of the cells had an affected cell cycle in the presence of SA-MIPs (Figure 8a). These results demonstrate the presence of an augmented sub-population of almost non-dividing cells in the total cell population as well as normally dividing sub-populations upon culture in the presence of MIPs (Figure 8). Based on the results from flow cytometry and fluorescence microscopy that not all cells became associated with SA-MIPs, it can be assumed that the slowly or non-dividing cells were cells that in some way had been engaged with the SA-MIPs. The average doubling time of the individually tracked cells can be estimated and was found to be roughly 14 h upon growth in a culture medium (Figure 8b).