Novocyte 2040r

After treatment, Raw264.7 macrophage cells were double-stained with Annexin V-fluorescein isothiocyanate and propidium iodide (PI) (FITC Apoptosis Detection Kit I; BD Biosciences, San Jose, CA, United States) according to the instructions. Quantification was then performed using a flow cytometer (NovoCyte 2040R; ACEA Bioscience, San Diego, CA, United States) and analyzed using NovoExpress 1.4.1 Software (ACEA Bioscience, San Diego, CA, United States).

Apoptosis was detected using Annexin V-FITC/PI cell apoptosis detection kit (C1063, Beyotime Biotechnology, Jiangsu, China). Cells from different groups were digested with trypsin without EDTA, resuspended in the binding buffer, stained with Annexin V-FITC for 15 min and PI for 5 min, and then analyzed by flow cytometry (Novocyte 2040R, ACEA, USA).

Flow cytometric analysis was performed to determine the effect of DHCR24 on cell cycle distribution. Briefly, cells grown in 6-well plates were treated with shRNA for 48 h. Then, cells were harvested and fixed in 75% ethanol solution. After centrifugation, cells were washed twice with cold PBS, then incubated with RNase A and stained with propidium iodide for 30 min in the dark. Cell cycle distribution was analyzed by flow cytometry (NovoCyte 2040R; ACEA Bioscience, Inc.; Agilent Technologies).

Cells from different groups were digested with trypsin without EDTA, resuspended in calcium‐enriched buffer, stained with Annexin V‐FITC for 5 minutes and PI for 15 minutes and then analysed by flow cytometry (Novocyte 2040R, ACEA, USA). Cell apoptosis was also determined with a Hoechst 33258 kit according to the manufacturer's instructions. The cells were observed under a fluorescence microscope (Olympus) and Hoechst 33258 was detected at an excitation wavelength of 340 nm. For each well, three visual fields were randomly selected. The number of positive‐staining HUVECs and total cells were counted and apoptosis was evaluated by the ratio of positively stained cells to the total number of HUVECs.

The intracellular ROS level within K. mikimotoi was measured using a ROS detection kit (Biyuntian, Shanghai, China) with slight modifications. The methods were as follows: (1) after 4, 8, 12, 24, 36, and 48 h of culture, 40 ml of algal solution was centrifuged at 4°C, and the supernatant was immediately discarded to collect algal cells; (2) DCFH-DA probe dye was added, and the mixture was incubated at 37°C for 30 min; (3) the mixture was centrifuged (at 1,000 rpm, 5 min), and the algal cells were washed with PBS; (4) the mixture was centrifuged again to settle the solution, and the supernatant was discarded to obtain the algal cells; (5) after resuspending the cells using PBS, the fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a flow cytometer (Novocyte 2040R, ACEA, United States).