*3.13. Flow Cytometry-Based Analyses*

In all flow cytometry-based analyses, cells (4 × 10<sup>4</sup> cells/mL) were incubated without (as a non-treated control, cells were in serum-free medium) and with 200 μg/mL SC, SCA, SCH, or SCAH (these fucoidan extracts were prepared as a 20 mg/mL stock solution by thoroughly dissolving fucoidan powder in PBS; a final concentration of 200 μg/mL was obtained by diluting the stock solution with serum-free medium) for 48 h, and then cells were de-attached by trypsin and rinsed two times in cold PBS to obtain cell samples. Then, each flow cytometry-based analysis was performed according to the protocols below.

For the MMP analysis, the assay was conducted according to the method of Yang et al. [22]. Briefly, single-cell suspensions were washed twice with PBS and incubated, in the dark, for 20 min at 37 ◦C with TMRE (100 nM). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution.

For Bcl-2 expression analysis, single-cell suspensions were fixed using fixation buffer at 37 ◦C for 20 min. Then, the cells were permeabilized using permeabilization buffer and incubated, in the dark, for 1 h at RT with FITC-labeled anti-Bcl-2 antibody (1:25, *v*/*v*). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution.

The analysis of cytochrome c release was conducted by following the method of Huang et al. [41]. Briefly, single-cell suspensions were fixed using fixation buffer at 37 ◦C for 20 min. Then, the cells were permeabilized using permeabilization buffer and incubated, in the dark, for 1 h at RT with FITC-labeled anti-cytochrome c antibody (1:10, *v*/*v*). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution.

For activated caspase -9 and -3 analyses, the method of Huang et al. was used [41]. Briefly, single-cell suspensions were incubated, in the dark, for 1 h at 37 ◦C with FITC/LEHD/FMK solution (for caspase-9 detection) or FITC/DEVD/FMK solution (for caspase-3 detection). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution.

The annexin V-FITC/PI staining analysis was conducted using an annexin V-FITC apoptosis detection kit according to the method of Yang et al. [22]. Briefly, single-cell suspensions were incubated, in the dark, for 15 min at RT with annexin V-FITC (1:20, *v*/*v*) and PI (1:20, *v*/*v*). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution.

For phosphorylated Akt and mTOR analyses, the assays of phosphorylated Akt and mTOR were conducted using the method of Huang et al. [76]. In brief, single0cell suspensions were fixed using fixation buffer at 37 ◦C for 1 h. Then, the cells were incubated, in the dark, for 1 h at RT with allophycocyanin (APC)-conjugated anti-Akt1 antibody (1:50, *v*/*v*), FITC-conjugated anti-phospho-Akt (Ser473) antibody (1:20, *v*/*v*), or phycoerythrin (PE)-conjugated anti-phospho-mTOR (Ser2448) antibody (1:20, *v*/*v*). After labeling, cells were washed and re-suspended for flow-cytometric measurement in staining solution. All of the flow-cytometric analyses described above were conducted using a BD Accuri C6 flow cytometer (San Jose, CA, USA).
