Flat bottomed polystyrene microplate

The evaluation of biofilm formation was carried out by determining the biomass and viability of each probiotic strain in the sessile phase. Overnight cultures of L. reuteri and B. longum strains in both MRSB and MRSB with 0.05% cysteine were standardized to a concentration of 5 × 10⁵ CFU/mL, placed in a flat-bottomed polystyrene microplate (Corning Inc., Corning, NY, USA), and incubated for 24–48 h at 37 °C under 5% CO₂ conditions. Then, the planktonic phase was gently removed, and the wells were washed with PBS (pH 7.4) 3 times. To assess the biomass of the strains, the microplate wells were dried, colored with 0.1% safranin, and washed again with PBS. The colored biofilm was resuspended in 30% acetic acid (v/v) and then OD₄₉₂ was measured by spectrophotometer. Control wells containing only the medium were included. The production of biofilm was obtained by comparing the OD value obtained from the arithmetic mean of the values of each strain with the cut-off value (ODc), defined as the average of the values that was obtained from the control. To assess the cell viability of the samples, microplate wells with adherent biofilm were scarified in PBS, serially diluted, and seeded on MRSA (Oxoid) plates. After an incubation period of 24 h and 48 h at 37 °C, CFU/mL were counted [63 (link),64 (link)].

To detect IgG against surface proteins of non-infected RBCs, each well of a 96-well, flat-bottomed, polystyrene microplate (Corning Incorporation, Corning, NY, USA) was coated with approximately one million RBCs (obtained from a healthy non-exposed individual whose blood type is O⁺) diluted in PBS containing 1 % (w/v) BSA (PBS/BSA), following an overnight incubation at 4 °C. After five washes with PBS the plate was blocked with 5 % BSA for 2 h at 37 °C. Plates were washed again and were incubated with sera samples in duplicate diluted 1:100 in PBS/BSA for 2 h at 37 °C. Wells were washed again and then incubated with HRP-conjugated anti-human IgG antibody (Sigma-Aldrich) diluted 1:500 in PBS/BSA for 90 min at 37 °C. The binding was revealed with OPD substrate as described in the section ‘Detection of total IgG’. The levels of specific IgG were expressed as reactivity index (RI), which was calculated as the ratio between the mean optical density (OD) by each sample duplicate and the mean OD plus three standard deviations of samples from 11 malaria-naïve volunteers never exposed to malaria.