Powerwave ht spectrophotometer

For the cell proliferation assay, cells were transfected with si-control and si-PD-L1 and then plated at 3 × 10³/well in 96 wells, and cell growth was determined using Incucyte™ for 72 h and the CellTiter 96 Kit (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega, Madison, WI, USA), as previously described in the manufacturer’s instruction. Afterward, 20 μl of the MTS solution was treated to each well, and the plates were incubated for another 2 h. The absorbance was measured at 490 nm with a PowerWave HT spectrophotometer (Biotek Instruments, Winooski, VT). Each experiment was conducted in triplicate.

For the cell proliferation assay, the cells were plated in 96-well cell culture plate (5 × 10³ cells) and cultured overnight prior to treatment with KML001. The effects of the treatments on cell growth were determined with CellTiter 96 aqueous nonradioactive cell proliferation assay kit (MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega, Madison, WI, USA) as described in the manufacturer’s instruction. Absorbance was measured at 490 nm with a PowerWave HT Spectrophotometer (Biotek instruments, Winooski, VT, USA). For trypan blue exclusion assay, cells were plated in 6-well plates (1.5 × 10⁵ cells/well) for 24 h, followed by KML001 treatment for 24 h. The cells were trypsinized, stained with trypan blue, and counted under the microscope as viable cells. Alternatively, after staining with trypan blue, cells were counted by countess^TM automated cell counter from Invitrogen (Carlsbad, CA, USA). Each experiment was performed in triplicate.

We developed an ELISA as an additional in vitro test for the diagnosis of LBRF. For this purpose, purified CihC, CihC-N, CihC-C, and GlpQ (1 ng/µl each), respectively were coated separately or in combination with GlpQ on a 96-well medium-bind microtiter plate (Greiner, Nürtingen, Germany) overnight at 4°C and afterward blocked with PBS containing BSA (0.45 %). The microtiter plates were air-dried and stored for several weeks at 4°C upon use. For each test, 100 µl of patient samples diluted 1:100 in sample dilution buffer (NovaTec Immundiagnostica GmbH) was added to the wells and incubated for 1 h at 37°C, respectively. The wells were then washed three times with NovaTec wash buffer and subsequently incubated with horseradish peroxidase-conjugated immunoglobulins (IgM, 1:50,000; IgG, 1:360,000) (NovaLisa conjugate, NovaTec Immundiagnostica GmbH) for an additional 30 min at RT. Subsequently, the wells were washed three times with NovaTec wash buffer and the protein complexes were detected by adding 100 µl TMB solution (NovaTec Immundiagnostica GmbH). The reactions were developed for 15 min at RT and terminated by adding 100 µl stop solution (NovaTec Immundiagnostica GmbH). The absorbance was measured at 450 nm and 620 nm as a reference using a PowerWave HT spectrophotometer (BioTek, Winooski, VT).

Approximately 50 mg of gut tissue was briefly homogenized using a Polytron PT1200 homogenizer in 0.5 mL of Trizol (Invitrogen, Carlsbad, CA, USA). Samples were sonicated for 30 s before 200 ul of chloroform were added, and samples were centrifuged for 15 min at 1200 rpm at 4 °C. The supernatant was transferred to microcentrifuge tubes, and samples were mixed with 500 ul of isopropanol and left on ice for 10 min to allow RNA precipitation. Samples were centrifuged for 15 min at 1200 rpm at 4 °C. The upper aqueous phase was discarded, and the pellets were washed with 1 ml of 70% ethanol, after which the samples were further centrifuged for 5 min at 7500 rpm at 4 °C. The supernatant was aspirated and pellets were air-dried for 15 min and then resuspended in 25 ul of RNase-free water. RNA concentration and quality were assessed by measuring the 260/280 nm ratio (> 1.8) using a Take3 micro-volume quantification plate (BioTek) and a powerwave HT spectrophotometer (BioTek). Total RNA integrity was determined by running RNA isolates on a 1% agarose gel stained with SYBR Green and verifying the bands for 28S and 18S ribosomal RNA.

The activity of the purified xylanases (Xyn11_Ec and Xyn11_Nb) was assayed at a range of high temperatures and alkaline pH. Enzymatic reactions at different temperatures (60, 70, 80, or 90 °C) were carried out by mixing 180 μL beechwood xylan 1% (Megazyme) with 20 μL of the enzyme in a Tris–HCl buffer (50 mM) at a pH of 9.0. The enzyme concentration was adjusted to assure a lineal response. Analyses at different pH values were carried out at 90 °C, using 50 mM buffered solutions at the following pH: 5.0 (acetate), 6.0 and 7.0 (phosphate), 8.0, 9.0 (Tris–HCl), and 10.5 (CAPS). Reactions were terminated by placing the tubes on ice. To measure sugar reduction resulting from xylan digestion, 100 μL of dinitro salicylic acid (DNS) solution was added to the reaction tubes, which were subsequently boiled for 10 min. After boiling, 900 μL of miliQ H₂O was added, and the tubes were centrifuged. A fraction of the supernatant (300 μL) from each reaction tube was transferred to a 96-well plate, and optical density at 540 nm (OD₅₄₀) was measured using a PowerWave HT spectrophotometer, from BioTek Instruments (Winooski, VT, USA). Statistical analysis was performed by two-way ANOVA, using a p value < 0.001.