Biosciences accuri c6

We evaluated the effects of cyclodextrins and miglustat on lysosomal changes as NPC manifestations using LysoTracker^® Green DND-26. WT and Npc1-null CHO cells were seeded at 1 x 10⁵ cells /well in 12-well plates and incubated at 37 °C for 24 h to allow cell adhesion. The cells were then treated with or without cyclodextrins and miglustat for 24 h. We confirmed that 8–24 h of treatment with HP-β-CD exerted sufficient and significant attenuating effects against NPC in our preliminary results (data not shown). The concentration ranges of cyclodextrins and miglustat were based on our previous studies [23 (link),28 (link)]. The cells were washed with phosphate-buffered saline and stained with 50 nM LysoTracker^® dissolved in DMEM/F12 (1:1) with 10% FBS and antibiotics for 15 min at 37 °C. Then, the cells were collected by trypsinization, and LysoTracker^® fluorescence was measured by a flow cytometer (BD Biosciences Accuri™ C6, Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) using a 488 nm laser. Finally, using a 530 ± 30 nm filter, fluorescence was detected in the FL1 channel. Data from 10,000 cells were collected and represented as the mean fluorescence intensity using BD Accuri C6 software (Becton Dickinson Biosciences). We performed three independent experiments (N = 3) for each group and show the mean value and standard error of the mean (SEM) of the three values.

Cell apoptosis ability was detected by Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (#556547; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with specification of manufacturer. After 48 hours transfection, cells were gathered and washed 3 times by phosphate-buffered saline (PBS). Then, we resuspended the cells into 1× binding buffer (1×10⁶ cells/ml). Next, cells were stained with Annexin V-FITC for 15 minutes and propidium iodide (PI) for 5 minutes in the dark before examined by flow cytometry (BD Biosciences Accuri C6; Becton, Dickinson and Company). And the results were analyzed by Flowjo software (Flowjo, Ashland, OR, USA). The experiment was repeated three times.