Cytexpert 2

For cell cycle analysis in HCT 116 and HT29 cells, 8 × 10⁴ cells were seeded and incubated overnight at 37 °C in a 5% CO₂ atmosphere. Following exposure to each compound for 24 h, cells were trypsinised and centrifuged (300 g, 5 min) to collect the cell pellets, which were then fixed with 70% ethanol on ice for 15 min. They were then centrifuged again to collect the pellets and incubated with staining solution [50 μg·mL⁻¹ propidium iodide (PI), 0.1 mg·mL⁻¹ ribonuclease A, 0.05% Triton X‐100] at 37 °C for 1 h, followed by analysis using CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). For the detection of apoptosis and cellular mitochondrial membrane potential (MMP) in HCT 116 cells, annexin V/PI double‐staining and JC‐1 staining methods were followed, respectively. Commercially available apoptosis detection kit and MitoScreen (JC‐1) kit were purchased from Becton Dickinson, North Ryde, NSW, Australia. Following the manufacturers' instructions, samples were analysed using the CytoFLEX or Gallios flow cytometer (Beckman Coulter) within 1 h of staining. Data were analysed using cytexpert 2.1 or kaluza v1.2 software (Beckman Coulter).

To assess the platelet activation, the CD62P expression and PAC-1 binding to platelets were determined as previously reported [34 (link)] with slight modification. Gel-filtered platelets (1 × 10⁶ cells per mL) prepared from the blood of healthy subjects were incubated with EPSp, IPSp, EPSpm, and IPSpm (5 mg/mL or 1 mg/mL, 4:1, v/v) or different controls (PBS, basal medium before and after fecal fermentation, 1:4, v/v) for 10 min at 37 °C, followed by co-incubation for another 30 min with fluorescein isothiocyanate (FITC)-conjugated anti-human CD62P and FITC-conjugated PAC-1 antibodies, respectively. Then, collagen was added to activate platelets for 1 min in the presence of 1 mM Ca²⁺. Samples were immediately fixed with 1% paraformaldehyde, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter Inc., Brea, CA, USA) within two hours. The data were collected and processed by CytExpert 2.0 software (Beckman Coulter Inc., Brea, CA, USA).

SLC5A8-pLVX cells were treated for 72 h with varying concentrations of 2,4,6-THBA. Adherent cells were collected by trypsinization and pooled with floating cells, washed with 1X PBS. Cell cycle analysis was performed by adding Vybrant^® DyeCycle™ Green Stain (Invitrogen, Carlsbad, CA, USA) at a final concentration of 250 nM to 1 mL cell suspension. The samples were analyzed by flow cytometry following incubation for 30 min at 37 °C. To detect apoptosis, cells were stained with an Annexin V/7-AAD kit as previously described [7 (link)]. All experiments were carried out by the CytoFLEX flow cytometer (Beckman Coulter, Miami, Indianapolis, IN, USA) using CytExpert 2.0 software (Beckman Coulter, Indianapolis, IN, USA).

Sublingual epithelial or subepithelial cells (5.0 × 10⁴) were added to conical bottomed wells of a 96-well plate. Ten μg/mL rat anti–human CD49f or isotype control in PBS + 1% BSA was added in a volume of 50 μL for 20 min on ice. Washing of the cells was followed by addition of 50 μL of 0.6 μg/mL Alexa Fluor® 647–conjugated goat anti–rat IgG (Biolegend) in PBS + 1% BSA for 20 min on ice. After two washing steps, cells were resuspended in 100 μL DPBS and analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Brea, CA, USA) using the CytExpert 2.0 software (Beckman Coulter). Singlets were gated in a forward scatter area and height plot, followed by discrimination of cell debris in a forward- and side-scatter plot. Expression of CD49f was assessed by measuring the relative fluorescence intensity for Alexa Fluor® 647.