Multiskan ascent microplate reader

In vitro interactions of PFA0660w with PfHsp70-x, and various Hsp constructs with ATS of PfEMP1 were examined by indirect ELISA. 100 ng each of purified bait proteins (recombinant purified PfHsp70-x or Hsp proteins) were coated on ELISA plates and blocked with 5% BSA in PBS overnight at 4 °C. The coated ligands were incubated with increasing concentrations of prey proteins (PFA0660w or ATS of PfEMP1 respectively; range: 10 ng to 500 ng) for 2 hrs at room temperature (RT), followed by extensive washing with 1X PBS. Washed plates were incubated with anti-PFA0660w-S (1:5000) or anti-ATS antibodies (1:5000) followed by anti-rabbit or anti-mice HRP conjugated secondary antibodies (1:10,000) for two hrs. Plates were developed using 1 mg/ml OPD (o-phenylenediamine dihydrochloride) containing H₂O₂, and absorbance measured at 490 nm (Multiskan Ascent Microplate Reader; Thermo Fischer Scientific).
For investigating protein-lipid interaction, a similar protocol as described above was followed where 100 ng of lipids were coated on ELISA plates and allowed to bind with varying concentration of hexahistidine tagged recombinant proteins (PFA0660w-C, PFA0660w-S, PfHsp70-x-C and PfHsp70-x-S). Bound proteins were detected by incubating plates with monoclonal anti-hexahistidine-HRP conjugated antibodies (1:10,000).

For quantification of VFAs, 0.5 g of thawed feces sample was combined with 8 ml of acid diluent (15 ml, 100 mmol/L of 2-ethylbutyric acid, and 50 ml, 5 mmol/L of hydrochloric acid) in a 20-ml centrifuge tube. After mixing by vortexing for 2 min, the solution was centrifuged at 10,000 × g and 4° for 20 min. The supernatant was transferred to a 10-ml centrifuge tube. A 1-ml supernatant was filtered by a 0.45-μm fiber filter into a sample bottle and then detected by Agilent 5975C gas chromatograph (Agilent, CA, United States) fitted with a flame-ionization detector. The samples (2 μl) were injected through the split injection port (50:1) onto a chromatographic column (PTX-Wax) (30.0 m × 0.25 mm column × 0.20 μm). The oven temperature was initially set at 60°C for 3 min and then increased at 10°C/min to 140°C and then held for 30 min. The detector temperatures were maintained at 300°C (Sato, 2009 (link)). The concentration of LA was determined by using a lactate assay kit (MAK064, Sigma-Aldrich, Darmstadt, Germany). A Multiskan Ascent microplate reader (Thermo Fisher Scientific, MA, United States) was used to detect the OD value at 570 nm.

Transwell cell migration was evaluated using 6.5 mm Transwell^® chambers with 8.0 μm pore polycarbonate membrane inserts (Corning, #3422). Quantification of migrating cells was performed 48 h after seeding by unstaining the chambers and measuring the absorbance at 570 nm in a MultiSkan Ascent microplate reader (Thermo Scientific, Budapest, Hungary) using the Ascent software.

Anti-BSA antibody levels were determined with ELISA. For this purpose, polystyrene 96-well plates were coated overnight at 4 °C with BSA (500 ng per well) diluted in 0.2 M carbonate buffer (pH 9.6). Each incubation step was preceded by three washes with PBST (PBS 1× + 0.05% Tween 20). The plates were blocked with a fat-free powder milk solution (5%) at room temperature for 2 h. Serial dilutions of the test sera were applied and incubated overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG (1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA), IgG1, or IgG2a were used for secondary labeling (2 h of incubation at 25 °C). The reaction was developed by adding a substrate solution of 0.3 mg/L ABTS and 0.1 M H₂O₂ followed by 50 min of incubation at 25 °C. OD_405nm values were measured in a Multiskan Ascent microplate reader (Thermo Fisher Scientific). The statistical significance (p-value) was determined using one-way ANOVA. The statistical analysis was performed using Statistica^® 12.7 (TIBCO Software Inc., Palo Alto, CA, USA).

Study subjects’ venous blood samples were collected after overnight fasting, centrifuged, and stored at −80 °C. High-density lipoprotein cholesterol (HDL-C) was determined using commercially available tests (D-HDL, Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) using the direct method. HDL-C was determined using the Siemens Advia 1800 analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) according to the manufacturer’s protocol.
For the determination of TrxR1 in the blood plasma, the human thioredoxin-1 ELISA Assay Kit (Prod. #RAB1756/Lot #0522F2032, Sigma-Aldrich, Inc., St. Louis, MO, USA) was used; for the determination of MPO in the blood plasma, the human myeloperoxidase ELISA Assay Kit, Item No. 501410, Cayman Chemical Company, Ann Arbor, MI, USA was used. The results were obtained using an Infinite 200 PRO multimode reader (Tecan Group, Mannedorf, Switzerland) and a Multiskan Ascent microplate reader (Thermo Labsystems, Helsinki, Finland). The procedures were performed according to the protocol of the ELISA kit manufacturer.
Concentrations of lipids, glucose, and other routine blood biomarkers were analyzed by standard methods.

96-well plates (Costar, Kennebunk, ME) were coated at 4 °C overnight with 100 μl/well of CAN1, CAN2 or CANB peptides or their respective fusion proteins at 1 μg/ml in 0.01 M bicarbonate buffer (pH 9.6). The plates were washed 5 times by PBS-T, followed by blocking with PBS containing 5% skim milk for 1 h at room temperature. The type/subtype specific universal antibodies was then added, followed by incubation for 1 h at room temperature before HRP conjugated goat anti rabbit IgG antibody was added. After incubation for 1 h at room temperature, the plates were washed 5 times before Tetramethylbenzidine (TMB) substrate was added for colorimetric development. Optical density at 450 nm was measured by a Multiskan Ascent microplate reader (Thermo Scientific, Shanghai, China). Unless specified, pre-immunization serum was used as negative control.

In 96-well plates, 500 cells per well for DAOY cells and 5 × 10³ cells per well for D283Med cells were seeded in sextuplicate. After 72 h of incubation at 37 °C, 0.5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma, St. Louis, MO, USA) was added, and after additional 3 h of incubation at 37 °C, the contents of the wells were removed, and formed crystals were resuspended in 150 μL DMSO per well for DAOY cells. However, for D283Med cells, 100 μL of isopropanol + 0.01 M HCl per well were added to the culture media, and the crystals were resuspended by pipetting up and down. In both cell lines, absorbance was measured at 570 nm in a MultiSkan Ascent microplate reader (Thermo Scientific, Budapest, Hungary) using the Ascent software. Cellular viability of the shERBB4 cells was calculated relative to the absorbance of control cells.