*2.8. Cell Uptake and Cell Tracking of PIPOx-FITC by Fluorescence Quenching Cytofluorometric Assay*

The uptake and intracellular tracking of PIPOx-FITC by RAW 264.7 cells was performed at 37 ◦C, for time period from 1–24 h, with 0.05, 0.1 and 0.5 mg/mL of PIPOx-FITC. The extracellular fluorescein isothiocyanate fluorescence of PIPOx-FITC has been quenched using 0.4% Trypan blue dye (Sigma-Aldrich, St. Louis, MO, USA). Trypan blue treated RAW 264.7 cells following exposure to PIPOx-FITC were assayed after 30 min incubation in dark by using immunocytometric evaluation. The amounts of adherent extracellular

and ingested intracellular PIPOx-FITC were distinguished and determined based on the difference between resulting total number of phagocyting cells and number of phagocyting cells following fluorescence quenching (Trypan blue assay).

#### *2.9. Colocalization of PIPOx-FITC in Macrophages*

The distribution fluorescently labeled PIPOx-FITC and its colocalization with specific organelles in RAW 264.7 macrophages was assessed by Confocal laser scanning microscope (CLSM) LSM510 META on an Axiovert 200 and 40×/1.2W C-Apochromat objective (Zeiss, Jena, Germany). The optical setup for FITC fluorescence was excitation with 488 nm laser line, and a 500–550 nm long-pass emission filter; for Mito-Tracker-Orange (Zeiss, Jena, Germany) a 543 nm laser line and a 565–615 nm long-pass emission filter, and for LysoTracker-deep red fluorescence. a 633 nm laser line and a 650–710 nm long-pass emission filter. Concentration of PIPOx-FITC for colocalization study was 1 mg/mL in full growth medium. Living cells were treated for 20 h with PIPOx–FITC, then rinsed with PBS and incubated for 10 min with MitoTracker or LysoTracker (both Molecular Probes, Eugene, OR, USA) at concentration of 75 × <sup>10</sup>−<sup>9</sup> mol/dm3. Cells were washed with PBS before imaging. A region of interest (ROI) analysis was performed using ZEN software (Zeiss) [40] The results are presented as the average of correlation coefficients from six different cells for each Tracker dye.

## *2.10. Statistical Analysis*

The immunobiological results were expressed as mean values ± SD. Data were tested for normality by Shapiro–Wilk test at the 0.05 level of significance. Statistics was performed by one-way ANOVA and post hoc Bonferroni test. Results were considered significant when differences equaled or exceeded the 95% confidence level (*p* < 0.05). Statistics was completed by ORIGIN 2018 software (OriginLab Corporation, Northampton, MA, USA). Pearson's correlation coefficient has been applied to compare the strength of the relationship between variables.
