Figure 3.

Pre-treatment with the probiotic Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri blocks internalization of BoNT/A into Caco-2 cells. (A) Caco-2 cells were treated with media (control) or with BoNT/A for 4 h at 37 °C. Some Caco-2 cells were either pre-treated with Lactobacillus acidophilus (LA), Lactobacillus rhamnosus (LGG), or Lactobacillus reuteri (Lr) for 30 min at 37°C at either high (10⁸ CFU) or low (10⁴ CFU) before addition of BoNT/A complex. Cells were fixed and stained with Alexa-488 labeled antibodies to BoNT/A, DAPI (nuclear), and Rhodamine-Phalloidin (actin cytoskeleton). Representative images at 40− magnification showing BoNT/A fluorescence are shown; (B) The cellular uptake of BoNT/A was quantified by determining the mean fluorescence of three randomly chosen optical fields from each of the four coverslips per strain per experiment using ImageJ software. Values represent means of four independent experiments ± SEM. Statistical significance was determined using two-way ANOVA followed by the Tukey-Kramer test where multiple groups are compared with p-values < 0.05 are taken to indicate significant differences between groups (*).