Epoch plate reader

Fecal samples were homogenized with RIPA buffer (1:2), and the proteins were obtained by centrifuging (10,000 rpm, 10 min, 4°C). Levels of S100B were measured with a DuoSet S100B kit (R&D Systems) by ELISA according to the manufacturer’s protocol. The absorbance (450 nm) was determined using an Epoch plate reader (BioTek). The range of S100B detection was 46.9–3,000 pg per mg of protein.
EGCs line (6×10⁵ cells/well) were seeded in six-well plates and treated with TcdA or TcdB for different times. EGC supernatants were collected (stored at -80°C until use) and centrifuged (10,000 rpm, 10 min, 4°C), and secreted S100B was measured with a DuoSet S100B kit (R&D Systems) by ELISA according to the manufacturer’s protocol. The absorbance (450 nm) was determined using an Epoch plate reader (BioTek). The range of S100B detection was 46.9–3,000 pg/ml.

The serum neutralizing antibody levels against SARS-CoV-2 were determined using a microneutralization test against the Washington strain and the Delta variant, as published previously [3 (link)]. Briefly, 200 TCID₅₀ of the SARS-CoV-2 test strain was incubated with two-fold dilutions of the serum samples in 96-well plates for 1 h at 37 °C. Vero 81 cells (2 × 10⁴) were then added to each well and incubated at 37 °C for 84 h. After 84 h, the cells were fixed by adding 200 µL of cold fixation solution (1:1 mixture of ethanol and methanol) to each well and incubating the plates at −20 °C for 30 min. Once the cells were fixed, the SARS-CoV-2 titer in each well was measured by quantitating the spike protein using a SARS-CoV-2-specific anti-S antibody in a standard ELISA format, and the absorbance at 405 nm was measured using a BioTek Epoch plate reader (Agilent Technologies Inc. (Wood Dale, IL, USA)). The highest serum dilution that resulted in a ≥80% reduction in absorbance when compared to the control samples (without neutralizing antibodies) was determined as the 80% microneutralization titer (MN₈₀).

The cytotoxicity
of the series of DHPs and their labeled homologues was evaluated in
human umbilical vein endothelial cells (HUVECs; American Type Culture
Collection, Manassas, VA, US) by adapting pre-established protocols.^{25 (link)} Cells were grown in Medium 199 (M199, LabClinics,
Barcelona, Spain) supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Invitrogen, US), 1% penicillin/streptomycin, and 10 mM
glutamine, seeded at a density of 2.2 × 10⁴ cells/mL
in MW96 plates and incubated for 24 h at 37 °C. Then, the medium
was replaced with samples diluted in M199 without complements and
plates were incubated for a further 48 h at 37 °C. The medium
was then replaced by 10% 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1, Roche, Penzberg,
Germany) in M199 and incubated under the same conditions for 4 additional
hours. The cleavage of the WST-1 tetrazolium salt to yield formazan,
occurring inside living cells, was spectrophotometrically determined
by measuring absorbance at 440 and 600 nm in an EPOCH plate reader
(BioTek, Agilent Technologies, Santa Clara, CA, US). Cells in the
medium without any treatment were included as the positive growth
control, cells in 50% dimethyl sulfoxide were used as the negative
control, and the maximum percentage of water added with the samples
(5% v/v) was also tested. Samples were assayed in triplicate.