Stat fax 4200 microplate reader

The VEGF concentrations were calculated by interpolation from a standard curve (0.001–1 ng/mL) performed with the mouse VEGF protein, VEGF mBA-165 (Cat# sc-4571, Santa Cruz Biotechnology, Santa Cruz, CA USA). Tumor protein was obtained and quantified through the same protocol implemented for cytokine determination.
The polystyrene wells (96-well plate, MaxiSorp Nunc Cat# NNC#442404) were coated with 50 µL of tumor protein (10 µg) or with the different concentrations of the standard curve, all diluted in bicarbonate buffer (pH 9.6), coated per duplicate, and incubated at 4 °C overnight. The plate was washed and blocked with 200 µL of PBS/bovine serum albumin (BSA) 1%/Tween 20 0.05% for 1 h at 4 °C and washed again. Furthermore, 50 µL of anti-VEGF/C-1 antibody (Cat# sc-7269, RRID:AB_628430, Santa Cruz Biotechnology) in a 1:200 dilution was added and incubated for 1 h at 4 °C. After washing, 50 µL of m-IgGκ/BP-HRP (Cat# sc-516102, RRID:AB_2687626, Santa Cruz Biotechnology) (1:400) was added and maintained for 2 h at room temperature. An enzyme-substrate reaction was developed with 50 µL of substrate solution and stopped after 15 min with 50 µL 2N sulfuric acid. The plates were read at a wavelength of 492 nm in a Stat Fax 4200 microplate reader (Awareness Technology). Cytokine and VEGF concentrations were calculated by interpolation from a standard curve.

Polystyrene wells (96-well plate, MaxiSorp Nunc Cat# NNC#442404) were coated with 50 μl of splenic protein (10 μg), sera (dilution 1:2), or standard curve (0.001-1 ng) with VEGF mBA-165 (Cat# sc-4571, Santa Cruz Biotechnology) in bicarbonate buffer (pH 9.6) per duplicate and incubated at 4°C overnight. The plate was washed and blocked with 200 μl of PBS/BSA 1%/Tween 20 0.05% for 1 h at 4°C. After washing, 50 μl of anti-VEGF/C-1 antibody (Cat# sc-7269, RRID:AB_628430, Santa Cruz Biotechnology) in a 1:200 dilution was added, followed by incubation for 1 h at 4°C. After washing, 50 μl of m-IgGκ/BP-HRP (Cat# sc-516102, RRID:AB_2687626, Santa Cruz Biotechnology) (1:400) was added and maintained for 2 h at room temperature. An enzyme-substrate reaction was developed with 50 μl of substrate solution and stopped after 15 min with 50 μl 2N sulfuric acid. The plates were read at a wavelength of 492 nm in a Stat Fax 4,200 microplate reader (Awareness Technology). Cytokine and VEGF concentrations were calculated by interpolation from a standard curve.
Cytokine and antibody determination were performed after proper ELISA standardization.

Genomic DNA was isolated from hepatopancreas samples using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s directions. The DNA purity was checked by UV absorption (260/280 nm, DS-11 Spectrophotometer, DeNovix, Wilmington, NC, USA) [7 (link)]. Total 5mC DNA level was quantified using the MethylFlash Global DNA Methylation (5mC) ELISA Easy Kit (EpiGentek, Farmingdale, NY, USA). This kit provides a fast and precise method to determine global DNA methylation changes, with a detection limit of 5 nanograms (ng) of fully methylated DNA. In addition, the use of such ELISA-based assays for serial measurements of 5mC can serve as a cost-effective and reliable alternative to commonly used methods for whole-genome DNA methylation quantitation, such as chromatography, radioactive filter-binding, or bisulfite sequencing-based methods [32 (link),33 (link)]. Optical density (OD) was measured at a wavelength of 450 nanometers (nm) with a Stat Fax 4200 microplate reader (Awareness Technology, Palm City, FL, USA). For absolute 5mC quantifications, a standard curve was created by graphing the corresponding ODs versus the log₁₀-transformed concentrations of the positive controls. The coefficients of determination (R²) for ELISA calibration curves were above 0.97; an example is shown in Figure 1.

Polystyrene 96-well plates were sensitized (Maxisorp Nunc) with 50 µl of protein (10 µg), serum (1:2 dilution), or VEGF (Santa Cruz Biotechnology) standard (0.001 ng-1ng) in bicarbonate buffer (in duplicate) and incubated at 4°C overnight following manufacturer instructions. Subsequently, the plates were washed three times with a wash solution. 200 µl of the blocking solution (1%) was added and incubated for one hour at 4°C. Next, 50 µl of anti-VEGF C-1 antibody (sc-7269, Santa Cruz Biotechnology) (1:200) was incubated for one hour at 4°C. For recognition of anti-VEGF IgG, 50 µl of m-IgGκ BP-HRP (sc-516102 Santa Cruz Biotechnology) was added (1:400) for 2 h at room temperature. Finally, after washing, the enzyme-substrate reaction was performed with 50 µl of the chromogen solution. The reaction was stopped after 20 min with 50 µl of 2 N sulfuric acid. The absorbances were determined at 492 nm in the Stat Fax 4200 microplate reader (Awareness Technology). All cytokine and VEGF concentrations were calculated with interpolation from a standard curve. The determination of TES concentration, dilutions of sera, and antibodies was developed after the standardization of the respective ELISAs.

Polystyrene wells (96-well plate, MaxiSorp Nunc Cat# NNC#442404) were coated with 50 µL of mice sera/bicarbonate buffer (pH 9.6) (1:1000) or with 1 µg/1 mL of 4T1 crude extract, overnight at 4 °C. The plate was washed three times with PBS/Tween 20 0.05% and blocked with 200 µL of 3% BSA and washing solution for 30 min at room temperature. The plate was washed as mentioned. For the anti-4T1 IgG determination, plates were incubated with mice sera (1:200) for 2 h at room temperature and washed. In both cases, 50 µL of peroxidase goat anti-mouse IgG (Jackson, PA, USA RRID:AB_2338511) at 1:10,000 dilution was added over 90 min at room temperature. An enzyme-substrate reaction was developed by adding 50 µL of freshly prepared substrate solution (0.05% o-phenylenediamine/0.01% H₂O₂/0.1 M sodium citrate/0.1 M citric acid) and stopped with 50 µL 2N sulfuric acid after 10 min. The plate was read at a wavelength of 492 nm in a Stat Fax 4200 microplate reader (Awareness Technology). Cytokine, VEGF, and antibody determination were performed after proper ELISA standardization.

Glutamate level was measured in retinal homogenate by using glutamate assay kit (Cat. number ab83389, Abcam), according to the manufacturer's instruction. Briefly, 50 μL of standards and retinal homogenate (equal amount) was loaded in a clear 96-well plate, followed by addition of 100 μL reaction mix solution having assay buffer, glutamate developer, and glutamate enzyme mix. The plate was incubated for 30 minutes at 37°C (protected from light) and was read at 450 nm in microplate reader (Stat Fax 4200 microplate reader, awareness technology, Palm City, FL). The concentration of measured glutamate was expressed as nmol/μL/μg of protein.

Genomic DNA was extracted from hepatopancreas and ovotestis with the GenElute™ Mammalian Genomic DNA Miniprep Kit (Merck KGaA, Darmstadt, Germany) as per manufacturer’s instructions. A DS-11 Spectrophotometer (DeNovix, Wilmington, USA) was used to evaluate DNA purity by determining a sample absorbance spectrum at 260 nm and 280 nm, and calculating the A₂₆₀/A₂₈₀ ratio. Genome-wide 5mC levels in DNA samples were assessed using the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (EpiGentek, East Farmingdale, NY, USA), as previously described (Georgescu et al., 2021 (link)). Optical density (OD) was measured at 450 nanometers using a Stat Fax 4200 microplate reader (Awareness Technology, Palm City, FL, USA). For absolute 5-mC quantification, a standard curve was obtained by plotting the various concentrations of the positive controls against the corresponding ODs.