Fc500 5 colour analyzer

The uptake of bodipy-labeled cholesterol loaded in either sHDL or sHDL-PEG by HepG2 cells was quantified by confocal laser scanning microscopy and flow cytometry. Briefly, liver hepatocellular carcinoma (HepG2) cells were cultured in Dulbecco’s Modified Eagle Medium with 10% FBS and 1% Pen Strep Glutamine and maintained in an incubator at 37°C and 5% CO₂. On day 0, 10⁴-10⁵ cells were seeded in a MatTek 35 mm petri dish in DMEM. The next day, media was aspirated and cells were washed with PBS. Fresh media containing sHDL and sHDL-PEG loaded with bodipy-labeled cholesterol was added at a final 22A concentration of 100 μg/ml. After incubation at 37 °C for 2 hours, cells were washed twice with PBS followed by fixation with 4% paraformaldehyde in PBS for 15 min at room temperature. 1% Triton-X solution was then added to the dish for 15 min and washed with PBS twice. Finally, the cell nuclei were stained with DAPI. The nuclei and bodipy fluorescence images were acquired on a Nikon A-1 Spectral Confocal microscope system (Nikon Corporation, Tokyo), with an excitation wavelength of 495 nm for bodipy. Quantification of cellular fluorescent signal was performed using a cell sorter (Beckman Coulter FC500 5-colour analyzer) at an excitation wavelength of 495 nm.

Cells were seeded into 35 mm petri dishes with coverslip attached (10⁵ /well) for confocal microscopy imaging, and into 6-well culture plate (2×10⁵/well) for flow cytometry studies. sHDL, PEG- sHDL, LIP, and PEG-LIP were added into wells at a DIO concentration of 5 μM, and incubated for 2 h at 37 ºC. Cellular uptake was visualized using a Nikon A-1 Spectral Confocal microscope system (Nikon Corporation, Tokyo) with an excitation wavelength of 488 nm. Quantification of cellular fluorescent signal was determined using a cell sorter (Beckman Coulter FC500 5-colour analyzer) at an excitation wavelength of 488 nm.
To investigate whether the uptake of HDL was mediated by the SR-BI, HCT 116 cells were pretreated with anti-SR-BI antibody (NB400-113, Novus biological) at a 1:100 dilution for 1 h at 37 ºC.^{24 (link)} The cells were washed twice with PBS and incubated with 5 μM DIO loaded sHDL for 0.5 h at 37 ºC, wahed with PBS and analyzed by flow cytometry.