Gen5 microplate reader version 3

The antibiotic sensitivity of L. reuteri PSC102 was determined against 14 antibiotics, including cephalexin, colistin sulfate, enrofloxacin, cefalonium, amoxicillin trihydrate, penicillin G procaine, norfloxacin, spectinomycin, tylosin base, cefuroxime sodium, florfenicol, penicillin G benzathine, gentamicin sulfate, and streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA), as previously described [26 (link)]. Briefly, 100 µL of cultured L. reuteri PSC102 was mixed with 100 µL of diluted antibiotic solution in a 96-well plate. After finally adjusting the concentration to 10⁶ CFU/mL, the bacteria were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by measuring the OD value at 600 nm using Gen5 microplate reader version 3.08 (BioTek, Winooski, VT, USA) and by streaking on Mueller–Hinton agar plate, respectively.

The proliferation of splenocytes was determined using the MTT assay (Zhang et al., 2020 (link)). The number of splenocytes was adjusted to 1 × 10⁶ cells/well in RPMI-1640 complete medium and seeded at 100 μL/well in a 96-well plate. Next, mitogen Con A and LPS (5 μg/mL) were added to each well to make the final volume of 200 μL, followed by incubation for 72 h at 37°C. The wells containing only RPMI-1640 medium without any mitogen were used as control. The MTT (5 mg/mL) solution (20 μL) was added to each well, followed by incubation for another 4 h. The medium was removed, and 200 μL DMSO was added to each well and shaken for 10 min in an orbital shaker. Finally, absorbance was measured at 570 nm using Gen5 microplate reader version 3.08 (BioTek, Winooski, VT, United States).