Gen5 reader control and data analysis software

IAP activity was determined using IAP and Bradford assay. The sample digests were added to the seeded 96-well plates and allowed a 4 h exposure at 37 °C, 5% CO₂. At the end of the 4 h exposure to digested control or test conditions, the wells were washed with 1× PBS, followed by the addition of 200 μL of 18 MΩ water. The plate was then subjected to sonication at 4 °C for 15 min. The samples were scraped off the bottom of the wells and subsequently used for the assay. A standard curve was prepared with dilutions of a standard solution of 0.25 mg/mL p-nitrophenol, prepared in p-nitrophenyl phosphate (pNPP). The assay was carried out by adding 25 μL of sample or standard solution in 85 μL of pNPP solution to a 96-well clear bottom plate, which was then incubated for 1 h at room temperature. For the Bradford assay, a standard curve was prepared using a 1 mg/mL bovine serum albumin (BSA, Rockland, NY, USA) solution prepared in 18 MΩ water. The experimental setup included a 5 μL sample or standard solution and 250 μL of Bradford reagent (Sigma) in a 96-well clear bottom plate. The plate was incubated at room temperature for 15–30 min in the dark. The IAP assay and Bradford assay plates were read at 405 nm and 595 nm, respectively, using the Synergy 2 plate reader, controlled by Biotek’s Gen5™ Reader Control and Data Analysis Software.

IAP activity was estimated through IAP and Bradford assays performed on the monolayers grown in 24-well plates. The wells were exposed to sample or control solutions for 4 h. At the end of the exposure period, the wells were washed with 1 × PBS and 200 μL of 18 MΩ water was added to each well. The plate was sonicated at 4°C for 15 min before transferring the content of each well into different 0.5 ml microcentrifuge tubes. For the IAP assays, 85 μL of p-Nitrophenyl Phosphate (pNPP, Sigma) solution and 25 μL of sonicated sample or IAP standard (prepared using p-nitrophenol and pNPP) was added to each well of a 96-well clear bottom opaque side plate and allowed to incubate at 37°C for 1 h. For the Bradford assay, 250 μL of Bradford reagent (Sigma) and 5 μL of sonicated sample or Bradford standard (prepared using Bovine Serum Albumin (BSA) and 18 MΩ water) was added to the wells. The Bradford assay plate was incubated at room temperature for 30 min. The IAP and Bradford assay plates were read at 405 and 595 nm, respectively, using Synergy 2 plate reader, controlled by Biotek's Gen5™ Reader Control and Data Analysis Software. Using the data, mg IAP/mg total cell protein was calculated and plotted.

B. infantis was grown in the absence and presence of 5 mg/mL GMO over a 72 h period under adhesion assay conditions. Aliquots of 150 μL of the bacterial suspensions were added to the individual wells of a 96 well microtiter plate. Other controls included a control containing no bacteria, and bacteria grown in deMan Rogosa Sharpe (MRS) (Difco, Sparks, MD, USA) broth supplemented with L-cysteine (0.05% w/v) (Merck). These experiments were performed in a concept 400 anaerobic chamber (Baker, ME, USA) and bacterial growth was monitored by determining OD_600nm using a Synergy-HT multidetector microplate reader driven by Gen5 reader control and data analysis software (BioTek Instruments Inc. Bedfordshire UK) at 0, 12, 24, 48 and 72 h. The microtitre plate was automatically shaken for 30 s prior to each measurement to achieve a homogenous suspension. The results are represented as the average OD_600nm of triplicate experiments performed on three separate occasions. The percentage increase in growth of B. infantis was calculated as the difference in OD_600nm between non-supplemented and GMO-supplemented bifidobacteria.

To estimate the amount of indole produced by E. coli under each condition, Kovac's Indole test was used. Post-exposure, 100 μL sample was collected from the apical chamber and added into a 96-well clear bottom plate after which, 150 μL of Kovac's reagent (Sigma) was added to each well. The plate was incubated for 30 min at room temperature and then read at 530 nm using Synergy 2 plate reader, controlled by Biotek's Gen5™ Reader Control and Data Analysis Software. An indole standard curve was prepared to estimate the amount of indole produced under different conditions.

The basolateral samples were subjected to charcoal treatment due to low glucose levels [23 (link)]. Activated charcoal (2% w/w) was added to the samples and mixed on a vortex for 30 s. The samples were then centrifuged for 30 s, and the supernatant was used for the assay. Prepared samples were added to glucose hexokinase liquid stable reagent (Infinity^TM) at a ratio of 1:150 in a 24-well clear bottom plate. Samples were incubated for 3 min at 37 °C, and the absorbance was read at 340 nm using a Synergy 2 plate reader, controlled by Biotek’s Gen5™ Reader Control and Data Analysis Software. The absorbance values along with the glucose standard (1 mg/mL in 0.1% benzoic acid) were used to calculate the amount of glucose (μg) per mL of each sample.

For IAP quantification, IAP and Bradford assays were performed on the cells grown in 24-well plates as previously described (Guo et al., 2017 (link)). The wells were washed with 1× PBS and 200 μl 18 megohm*cm deionized water was added to each well. The plate was sonicated at 4°C for 5 min before transferring the content of each well into different 0.5 ml microcentrifuge tubes. For IAP activity, 85 μl of pNPP solution was added to the required number of wells of a 96-well clear-bottom opaque side plate. To this, 25 μl of prepared samples and IAP standards (prepared using p-nitrophenol and pNPP) were added and allowed to incubate at 37°C for 1 h. For the Bradford assay, 250 μl of Bradford reagent was added to the required number of wells of a 96-well clear-bottom opaque side plate before transferring 5 μl of prepared sample or standards to those wells. The Bradford assay plate was incubated at room temperature for 30 min. The IAP and Bradford assay plates were read at 405 nm and 595 nm, respectively, using a Synergy 2 plate reader, controlled by Biotek's Gen5™ Reader Control and Data Analysis Software.