Elyra ps 1 laser scanning confocal microscope

The amine-modified MPI was labelled with 6-[fluorescein-5(6) carboxamido] hexanoic acid (FITC) by using similar PCA coupling protocol but with the PCA exchanged for a molar quantity of the probe. This coupling results in the formation of FITC modified inulin particles (MPIEDFITC). Then RAW 264.7 cells of 1 × 10⁵ cells/well were plated into each 12-well plate at 37 °C, 5% CO₂, high glucose media and left overnight to allow for proper attachment. After this, the cells were then exposed to FITC labelled MPI (MPIEDFITC) and FITC alone at a concentration of 20 µg/mL for 0.5, 2 and 4 h time point. At each time point, the cells were washed 3× in sterile PBS and then scraped off and spun down at 1000 rpm for 5 min. The cell pellet obtained after this procedure was then resuspended in 100 µL of PBS. To ensure an even and uniform preparation of cells before staining, the cells were spun down at 1000rpm for 5 min in a Cytospin4 cytocentrifuge (Thermoshadon, Cheshire, UK). Slides were then left to dry for 24 h before fixation for 10 min in neutral buffered formalin. Counterstaining and coverslipping were then performed using 4, 6 diamidino-2-phenylindole (DAPI) containing fluorescent mounting media. Images of each time point and treatment were then taken using a Zeiss Elyra PS.1 Laser Scanning Confocal Microscope with Zeiss ZEN lite software (CLSM).

The uptake of particles by RAW 264.7 cells was visualized using confocal laser scanning microscopy (CLSM; Zeiss Elyra PS.1 laser scanning confocal microscope, Germany). Briefly, cells were incubated overnight prior to being exposed to free Nile red, Nile red–loaded lipid micro-droplets and ILH at a Nile red concentration of 50 µg/mL for 1 h. After incubation and prior to staining, the nuclei and cytoskeleton with 4′,6-Diamidino-2-Phenylindole (DAPI) and Alexa-488, respectively, the cells were rinsed with PBS three times and fixed with paraformaldehyde (PFA) (4%) for 20 min. DAPI with the emission wavelength of 461 nm (excitation wavelength 358 nm) and Alexa- 488 with 525 nm emission wavelength (excitation wavelength 490 nm) appeared as blue and green, respectively.