Powerwave xs2 plate reader

Beta-galactosidase assays were performed by the method of J. Miller [12 ]. The following solutions were mixed together prior to performing the assay: 100× Mg²⁺ solution containing 0.1 M MgCl₂ and 4.5 M β-mercaptoethanol, 1× ONPG solution containing 4 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG) dissolved in 0.1 M dibasic sodium phosphate buffer pH (7.5). 3 µL of 100× Mg²⁺, 66 µL of 1× ONPG, 30 µL of cell lysate, and 201 µL of 0.1 M sodium phosphate were mixed together to initiate the reaction. Reactions were incubated at 37 °C until a faint yellow color developed. Reactions were stopped by adding 500 µL of 1 M Na₂CO₃. To determine beta-galactosidase activity, absorbance was read at 420 nm using a BioTeK Powerwave XS2 plate reader.

To determine antigen level in gradient fractions a non-competitive sandwich ELISA was used to measure PV3 D and C antigen content [38 (link)]. Briefly, two-fold dilutions of antigen were captured using a PV3-specific polyclonal antibody, and detected using PV3-specific, D antigen (Mab 1050) or C antigen (Mab 517.3) specific monoclonal antibodies (kindly provided by NIBSC), followed by anti-mouse peroxidase conjugate [39 (link),40 (link)]. To determine the presence of individual antigen sites concentrated samples were captured using a PV3-specific polyclonal antibody and detected using antigen site-specific monoclonal antibodies, kindly provided by NIBSC, followed by anti-mouse peroxidase conjugate. The specific antigen sites are listed in Table 1. BRP (Sigma) was used as the standard for D antigen content in each ELISA. All ELISAs were analysed through Biotek PowerWave XS2 plate reader.

Measurements of plasma levels of leptin were performed at room air (21% O₂) in anesthetized rats. After general anesthesia as described above, whole blood samples were taken through a cardiac puncture. Blood samples were drawn into collection tubes containing the anticoagulant EDTA (Sigma-Aldrich, USA) and kept on ice. After centrifugation, the plasma was stored at−80°C for leptin analysis by ELISA kit (#ab100773, Abcam, USA), an in vitro enzyme-linked immunosorbent assay for the quantitative measurement as previously described (Panetta et al., 2017 (link)). The assay was read using a power wave XS2 plate reader (Biotek Instruments, USA).
To confirm whether the chronic activation of leptin signaling pathways played a part in the HVR, subcutaneous injections of leptin (60 μg/kg) or equal volume of vehicle (saline) were carried out once daily for 7 days in CBI LZRs (n = 8 for each group), and breathing parameters were measured after 7 day injections during exposure to room air or hypoxia. To further confirm the CB's role, subcutaneous injections of leptin or saline were performed 7 days after the carotid sinus nerves were sectioned in each group (n = 8 for both).

The TP content in the extract was determined using the Folin-Ciocalteu method, as described by Kalita et al. [28 ]. To measure the TP, 30 μL of the extract was mixed with 50 μL distilled water in wells of a 96-well plate. Fifty microliters of Folin Ciocalteu reagent and 80 μL Sodium carbonate (75 g/L) was added to each well in the plate, mixed well with a pipette, and then shaken for 4 min in a plate reader. The plates were incubated for 2 h at 25 °C in the dark. The absorbance of the contents was measured with a Power wave XS2 plate reader (BioTek) at 760 nm. Gallic acid was used as the standard and TP were quantified as μg/g of gallic acid equivalent per gram freeze-dried sample.
To measure TF (30 μL) was added to 80 μL aluminum chloride (20 g/L) in a 96-well at-bottom microplate on ice. Samples were shaken for 30 sec. and then the plates were kept in the dark at 25 °C for 1 h. The absorbance of the reaction was measured at 415 nm. Quercetin was used as the standard. TF were expressed as μg of quercetin equivalents per gram of freeze-dried weight.