Costar 96 well plate

Total protein content was quantified with a Pierce™ Modified Lowry Protein Assay Kit (Thermo Fisher Scientific). For this, 2 µL of pre-gel solution was diluted in 38 µL of 1x DPBS and transferred to a well in a non-adhesive Costar^® 96-well plate (Corning Inc., Kennebunk, ME, USA). Next, 200 µL of modified Lowry protein assay was added per well before incubation at RT for 10 min. Fresh 1 N Folin-Ciocalteu’s phenol reagent was prepared by diluting 2 N Folin-Ciocalteu’s phenol reagent with an equal volume of Milli-Q^® water and 20 µL of this solution was added per well and incubated at RT for 30 min. The absorbance was read at 750 nm with Benchmark Plus™ microplate spectrophotometer system (Bio-Rad, Hercules, CA, USA). The protein concentration was determined based on a calibration curve derived from a dilution series of bovine serum albumin (Thermo Fisher Scientific). DPBS served as absorbance blanks. The protein concentration (µg/mL) from each organ-derived ECM was calculated from four independent experiments each performed in triplicate (Figure 1d).

Dual-luciferase assays were carried out as described with slight modifications (Hellens et al., 2005 (link)). Briefly, after 8 hours of induction, the transfected protoplast suspension was transferred to a 1.5 mL centrifugation tube and centrifuged at 100 g for 10 min. The supernatant was discarded and the pellets were re-suspended in 100 μL of 1X passive lysis buffer (PLB) provided in the Dual Luciferase Reporter Assay System kit (Promega). Protoplasts were disrupted by vortex for 10 s followed by centrifugation at 10,000 g for 2 min. A 5 μL of the supernatant sample was loaded into a well of a white flat bottom Costar 96 well plate (Corning). Dual-luciferase assays were performed in Synergy™ HTX Multi-Mode Microplate Reader (BioTek). A 40 μL luciferase assay reagent and a 40 μL Stop and Glo reagent (Promega) were injected per well. The ratio of LUC to REN was measured to represent the activity of the corresponding promoter when the effector plasmid DNA was co-transfected.

The reporter Luc constructs pCS001 (pGREEN-0800-LUC), pCS003 (pGREEN-800-TMMproLUC), and pRJH68 (pGREEN-800-SCRMproLUC) and the effector constructs pLJP152 (35Spro::SPCH), pLJP151 (35Spro::MUTE), and pMK165 (35Spro::SCRM) were published previously (10 (link), 11 (link), 42 (link)). The reporter and effector constructs were transformed into 5-wk-old N. benthamiana leaves via agroinfiltration as previously described (11 (link)). Six days after infiltration, tobacco leaves were harvested and assayed using the Dual-Glo Luciferase Assay System kit (Promega, E2920). The tobacco leaves were snap-frozen by liquid N₂, powdered, and subsequently mixed with 75 μL of the passive lysis buffer provided in the kit. The cellular debris was pelleted by centrifugation at 8,000 × g for 1 min. The supernatant was transferred into a well of a white flat-bottom Costar 96-well plate (Corning). An equal volume of Dual-Glo Reagent was added to each well. First, firefly Luc activity was measured. A volume of Dual-Glo Stop & Glo Reagent equal to the original tissue lysate was added to each well, and Renilla Luc activity was subsequently measured. The assay was performed using a GloMax 96-microplate luminometer (Promega).

Indole concentrations in the stools were determined using the hydroxylamine indole assay (15 (link)). Briefly, 250 mg of stool was suspended in 750 µl of 70% ethanol, vortexed for 30 s, and incubated in a 70°C water bath for 10 min. The samples were vortexed again for 30 s and centrifuged at 14,000 × g for 20 min at 40°C, and the supernatants were carefully pipetted. For the indole test, 100 µl of the supernatant was added in triplicates to a Costar 96-well plate (Corning, NY) containing 25 µl of NaOH (5.3 M) and 50 µl of 0.3 M hydroxylamine hydrochloride (NH₂OH-HCl) and incubated for 15 min at room temperature. Following the incubation period, 125 µl of H₂SO₄ (2.7 M) was added, thoroughly mixed, and incubated at room temperature for 2 to 30 min. Absorbance at a 530-nm wavelength was measured using a Spectramax I3 spectrophotometer (Molecular Devices, Sunnyvale, CA). The concentration of indole in the sample was determined using a standard curve obtained from known indole concentrations.

An adenosine triphosphate (ATP) assay was performed using the CellTiter-Glo 3D Cell Viability kit (Promega, Madison, WI). Each retinal organoid was placed in an individual well of a white Costar 96-well plate (Corning) with 100 µL of media. To each sample, 100 µL of reagent was applied, and the samples were incubated for 30 minutes at room temperature on an elliptical shaker under dark conditions. Luminescence readings were taken using a FLUOstar Omega Plate Reader (BMG Labtech, Ortenberg, Germany). ATP levels for each retinal organoid were determined from an ATP dilution series after being normalized to media-only controls. Data are presented as percentage ATP levels relative to the average ATP levels of four untreated retinal organoids. All treatment groups were n = 4. The normal distribution and variance of data were confirmed (Brown–Forsythe and Bartlett's tests), and a one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons was performed on the dataset. For correlation of retinal organoid size with ATP levels, retinal organoid sizes were determined by drawing around a standardized image and using the area measurement function within ImageJ (National Institutes of Health, Bethesda, MD). Area measurements were plotted against ATP levels, and a Pearson r-test was performed.

To assess drug efflux rate in human HCC cells, the Vybrant multidrug resistance assay kit (Molecular Probes #V13180, Eugene, OR) was used. The assay uses non-fluorescent calcein acetoxymethylester (calcein-AM) as a drug-mimic and a substrate for cancer cell efflux pumps. Calcein-AM is highly lipid soluble and permeates the cell membrane where it is converted to a fluorescent calcein by the intracellular esterases. The amount of intensely fluorescent calcein that is retained, can be measured as a measure of dye effluxed or an indication of dye retention inside the cell. The assay was performed as per the manufacturer’s protocol with some necessary optimizations. Briefly, the GH treated cells were counted and seeded at 50,000 cells/well in a black, clear bottom Costar 96-well plate (Corning #3603, Corning, NY) and then calcein-AM was added at a final concentration of 2 uM, and incubated at 37C for 2 hr. After thorough washing, fluorescence was measured at 494 (excS)/517 nm (emi) in a spectramax M2 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) and SoftMax Pro v6.2.1 software. Experiments were done in quadruplicate.