Biotek synergy 2 multi detection microplate reader

Radical scavenging activity of the DPPH radical extract was determined using a modified version of the Brand-Williams method [38 (link)]. 10 μL methanolic solution of diluted samples or Trolox (Sigma-Aldrich, Taufkirchen, Germany) solution in concentrations of 100 μM to 1.56μM were added to 190 μL of DPPH solution (Sigma-Aldrich, Taufkirchen, Germany) in a 96-well plate. The final concentration of DPPH was 0.1 mM in methanol. The solution was mixed gently and incubated for 30 min at room temperature in the dark. Subsequently, the absorbance against methanol was determined with a microplate reader at 517 nm (BioTek Synergy 2 Multi-Detection Microplate Reader, BioTek Instruments Inc., Winooski, Vt, USA). The DPPH radical scavenging activity of the extracts was calculated from the standard curve of Trolox and expressed as micromoles of Trolox Equivalents (TE) per gram of sample (μmol TE/g). Solvent controls containing only 10 μL methanol instead of samples and a positive control containing Trolox were used. Pure methanol was used as a blank. The inhibition of the DPPH radical by the samples was calculated according to the following formula: $DPPH\mathrm{scavenging}\mathrm{activity}\%=(1-\frac{A_{sample}-A_{blank}}{A_{control}-A_{blank}})\times 100(1-\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{blank}}}{{\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{blank}}})$

Total RNA was isolated from spleen tissue of zebrafish and ZF4 cells with Trizol reagent (TaKaRa, Tokyo, Japan) following the manufacturer’s protocol. The extracted RNA was re-suspended in 30 μl RNase-free water then quantified with a BioTek Synergy™2 Multi-detection Microplate Reader (BioTek Instruments, Winooski, VT) and agarose gel electrophoresis. One microgram of total RNA was used for reverse transcription with Revert Aid™ Reverse Transcriptase (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. The synthesized cDNA was stored at -20°C.

Gravimetric soil moisture and pH (using a ratio of 2 g soil to 4 mL DI H₂O) were assayed based on standard methods [9] (link). For total organic C analysis, carbonate (inorganic C) removal was first performed on dried, ground soils [9] (link). 50 mg of these processed soils were packed into tin capsules; %C and %N were determined using a Thermo Finnigan EA 1112 Series Flash Elemental Analyzer (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) [31] . Bio-available P concentrations were measured on air-dried and sieved soil (2 mm×2 mm) by extracting 3–5 g of soil with 0.5 M sodium bicarbonate for 30 minutes [32] . Extracts were filtered and analyzed colorimetrically using the ammonium molybdate-malachite green method [33] adapted for microplate analysis. NH₄⁺ and NO₃⁻/NO₂⁻ extractable N were analyzed from soils using 2M KCl with 1 hour shaking and a 22 hour extraction period [34] . This analysis was performed on soils that were frozen at −80°C. Although not fresh samples, these soils typically withstand extreme fluctuations in temperature [35] (link) and the data presented here are intended for within study comparison only. NH₄⁺ and NO₃⁻/NO₂⁻ were measured on a Lachat QuikChem 8500 Flow Injection Analyzer (Lachat Instruments, Hach Company, Loveland, CO) and BioTek Synergy 2 Multidetection Microplate Reader (BioTek, Winooski, VT) respectively.