Infinite f50 microplate reader

Serum IgG autoantibodies against double-stranded (ds)DNA were measured by ELISA. Plates were coated with 0.1 mg/mL salmon sperm DNA (ThermoFisher Scientific, Waltham, MA) in PBS in a warm room overnight and then rinsed with PBS the next day. Plates were blocked with 1% BSA in PBS for 1 h at 37°C. Samples were added at a 1:100 dilution and 6 subsequent two-fold dilutions to 1:3200, and incubated for 2 h at 37°C. After washing, samples were incubated with an alkaline phosphatase conjugated goat anti-mouse secondary antibody (Southern Biotech) and incubated for 1 h at 37°C. The plate was then washed, developed with phosphatase substrate (Sigma Aldrich) in 1M diethanolamine, pH 9.8 (Sigma Aldrich), and read at 405 nm using an Infinite F50 microplate reader (Tecan Group, Switzerland). Absorbance is reported as (optical density of sample/optical density of positive control 20-week old MRL/lpr serum on the same plate) x 100. Proteinuria was assessed semiquantitatively via Uristix where +1 is 30 mg/dL, +2 is 100 mg/dL, +3 is 300 mg/dL, and +4 is 2,000 mg/dL (Siemens Healthcare, Tarrytown, NY).

At the time of the study, serological testing for anti-echinococcal antibodies was performed at the Institute of Medical Microbiology and Hygiene at Ulm University Hospital. Echinococcus IgG was measured using the Cellognost^® Echinococcus IHA screening test (Siemens Healthcare Diagnostics GmbH, Marburg, Germany). Results were interpreted as positive if titers were >1:32. For this study, Echinococcus IHA results were recorded as semiquantitative values. Additionally, the Em2⁺ ELISA (Bordier Affinity Products SA, Crissier, Switzerland) was performed and interpreted qualitatively as positive according to the manufacturer’s instructions if the index $\frac{absorption of patient serum sample}{absorption of cut-off control}$ was ≥1.0. All ELISA washing steps were performed with an automated microplate washer (Biochrom Ltd., Cambridge, UK), and absorbance measurements were carried out on an Infinite F50 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). All patient sera were routinely further analyzed for their total IgE levels, using an electrochemiluminescence immunoassay on a Cobas e801 platform (Roche Diagnostics Deutschland GmbH, Mannheim, Germany), their Echinococcus-specific IgE fraction, and the eosinophilic cationic protein (ECP) using the ImmunoCAP²⁵⁰ (Phadia AB, Freiburg, Germany).

The main instruments were as follows: IKA-T10 basic homogenizer (IKA, Staufen, Germany); low-temperature high-speed centrifuge R134A (Eppendorf, Hamburg, Germany); Infinite F50 microplate reader (Tecan Group Ltd., Mannedorf, Switzerland); multifunction electrophoresis apparatus (Liuyi Instrument Factory, Beijing, China); Ice Maker 2BE-70-25 (ZIGERA, Germany); horizontal shaking table (GFL, United States); cassette and X film (Kodak, Japan).

Aseptically collect 1 g of chyme from the cecum and colon, and add an appropriate amount of PBS (pH = 7.4) to a 2 mL Eppendorf tube. Quickly freeze and store the sample in liquid nitrogen. After thawing the chyme, homogenize it thoroughly using a homogenizer. Centrifuge the homogenate for 20 min at 3000 rpm, and collect the supernatant. The concentration of total SCFA in the cecum and colon samples of each group was detected using an Infinite F50 microplate reader (Tecan, Switzerland) and an enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Jingmei Biotechnology Co., Ltd., Yancheng, China) for goose SCFA.

In order to discern the host specificity of phage, spot test and liquid assay were performed. The phage was tested against different clinical isolates of K. pneumoniae (BC936, E474, U2016, BC1415, BC1994) and E. coli (U3790, U3176, U1007, U1024, U2354) obtained from Sundaram Medical Foundation, Chennai, India. In addition, the phage was also tested against reference strains of E. coli (MG1655), Acenitobacter baumannii, Enterobacter cloacae, Pseudomonas aeruginosa and Enterococcus faecalis. Spot assay was done as reported earlier (Zhao et al. 2019 (link)). 300 µl of fresh bacterial culture was added to soft agar, overlaid on nutrient agar plate and allowed to solidify. 5 µl of the purified phage lysate (10¹² PFU/ml) were spotted on to the plate and incubated at 37 °C overnight. For liquid culture based evaluation, 180 µl of culture (0.05 OD) was mixed with phage (10⁸ PFU/ml) and incubated at 37 °C (Xie et al. 2018 (link)). The bacterial growth was monitored by measuring absorbance at 595 nm at 0, 30, 60, 90, 120 and 240 min using Tecan Infinite® F50 Microplate reader (TECAN, Männedorf, Switzerland) and the growth curve was plotted. The experiment was carried out in triplicates.

The activity of phages in combination with various antibiotics—Streptomycin, meropenem, colistin, erythromycin, ciprofloxacin and tobramycin, were tested by microbroth dilution method. The antibiotics were serially diluted from 64 µg/ml to 1 µg/ml and 10⁸ PFU/ml of KpG phages were added onto the wells. 0.05 OD (~ 10⁶ CFU/ml) of MTCC KP was inoculated and incubated overnight at 37 °C. After incubation, optical density at 595 nm was measured using Tecan Infinite® F50 Microplate reader. The ideal combination of phage and antibiotic, which effectively inhibited bacterial growth was selected and time kill study was performed as mentioned earlier. All experiments were performed in triplicates.