Ctb fitc

Cells were treated identically to those used for SPT, followed by fixation with 1% paraformaldehyde (PFA) in PBS at 4°C for 60 min. The fixed cells were washed with PBS twice, and 2 × 10⁵ fixed cells from each treatment were re-suspended in 200 μL PBS. The cells were labeled with Cholera toxin B FITC (CTB-FITC, 5 μg mL⁻¹; Sigma-Aldrich, Oakville, Ontario, Canada) and TS2/4-Cy5 (500 ng mL⁻¹) at room temperature for 10 min. Labeled cells were washed twice with PBS, re-suspended in 1 mL PBS, and transferred to a 24-well cell culture plate containing a coverglass (poly-L-Lysine treated). The plate was spun at 400 g for 7 min, and the well was washed 3 times with PBS. The coverglass was transferred to a microscope slide and sealed with Cytoseal 60, followed by imaging using TIRF. More than three independent labeling samples were imaged for each treatment.
TIRF imaging for cluster analysis was performed using an identical protocol as described above, with a lower concentration of the TS2/4-Cy5 conjugate (80 ng mL⁻¹). Fifteen cells were chosen for analysis based on DIC (the cells were apparently healthy and round) and staining (TIRF image showed TS2/4 labeling on the whole cell). Images of individual cells were processed in ImageJ by applying a threshold to identify labeled pixels and processed using the analyze particle function to measure clusters larger than 4 pixel² (0.07 mm²).

The protocol to this method is similar as for CTX (above). Instead of unlabeled CTX, 1.25 μg/mL of CTB-FITC (Sigma, Israel), CTB labeled with a fluorescent fluorescein derivative, was incubated with the appropriate concentration of plant extracts (2.5–0.075 mg/m, serial dilutions) in a total volume of 200 μL. After 2 h incubation at room temperature, wells were washed three times with PBS. Next, 200 μL of PBS was added and the intensity of fluorescence was measured at 490 nm excitation and 525 nm emission, using an Infinite M1000 PRO plate reader (Tecan). Each assay was repeated six times.

Cells were digested with trypsin, washed twice with PBS, fixed with 4% fresh paraformaldehyde, blocked with 1% BSA in PBS, and incubated with fluorescein isothiocyanate-conjugated Cholera Toxin B subunit (CTB-FITC, #C1655, Sigma-Aldrich) at 37°C for 30 min. Fluorescence signals were detected and analyzed using flow cytometry (ACEA Biosciences, San Diego, CA, USA).