Nanodrop nd 1000 uv vis

Vero cells were infected with SARS-CoV-2 (MOI 0.01) for 12 h, then treated with rhein at 50 and 200 μg/mL. After 2 h of incubation, RNA was collected by TRI Reagent (T9424, Sigma-Aldrich) and quantified by NanoDrop ND-1000 UV-Vis (Thermo Scientific, Waltham, MA, USA). m6A levels were analyzed by m6A RNA methylation assay kit as already reported [49 (link)].

DNA was extracted from whole body tissue of 89 adult P. confusa collected across 15 sites, and from abdomenal material of 87 adult A. urticae (one butterfly individual per nest) collected across 8 sites spread across the latitudinal gradient using the NucleoSpin^® 96 Tissue kit (Macherey–Nagel) (Figure 1). After extraction, the DNA samples were quantified and assessed using a spectrophotometer (NanoDrop^® ND-1000 UV-Vis; Thermo Scientific) and we measured concentrations of about 30 ng/µL.

Frozen bodies or heads were lysed in McIlvaine’s buffer (0.1 M citric acid, pH 4.2, and 0.2 M Na₂HPO₄, 29:21, v:v) and protein concentration was determined by NanoDrop^® ND-1000 UV-Vis (Thermo Fisher scientific, Waltham, MA, USA). Tissue homogenates containing 100 µg of protein were incubated at 37 °C with 8 µM of N-[6-[(7-Nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-glucosylceramide (C6-NBD-GlcCer) (Avanti Polar Lipids, Alabaster, AL, USA) in a final volume of 50 µL McIlvaine’s buffer for 1 h. Reactions were terminated by addition of three volumes of chloroform:methanol (2:1). Lipids were extracted and the lower phase was separated by TLC, as described under 2.13. N-[6-[(7-Nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-Ceramide (C6-NBD-Cer) was identified with an authentic standard (Matreya LLC, State college, PA, USA), using Amersham imager 600 (Amersham, Buckinghamshire, United Kingdom).

Total RNA was isolated from powdered roots according to Woodrow et al. (2016 (link)). RNA quantity and quality were determined spectrophotometrically using the NanoDrop ND-1000 UV-VIS (Thermo Scientific, Wilmington, MA) and separated on 1.5% agarose gel stained with SYBR safe (Invitrogen). mRNA was purified from ~500 μg of total RNA using a mRNA Isolation Kit (Roche) following manufacturer's instructions. First strand cDNA was synthesized from 1 μg of mRNA by reverse transcriptase with both random hexamer primers and anchored oligo dT according to the instructions of the SensiFAST^™ cDNA Synthesis Kit (Bioline).

Total RNA was isolated from frozen tissues and PAds-derived primary cells using Trizol reagent (Invitrogen, ThermoFisher Scientific, Monza, Italy) following the manufacturer’s instructions. RNA was quantified by spectrophotometry at 260 nm (NANODROP ND-1000 Uv/Vis, Thermo Fisher Scientific, Whaltham, MA, USA) and DNA contamination was removed by DNase I (Life Technologies, ThermoFisher Scientific, Monza, Italy) treatment. Reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) with a starting amount of 300 ng of digested RNA.
Real-Time PCR was conducted on a StepOne Plus System (ThermoFisher Scientific, Monza, Italy) using the following Taqman gene expression assays: SOX2 (Hs01053049_s1), OCT4/POU5F1 (Hs00999632_g1), NANOG (Hs02387400_g1), AXIN2 (Hs00610344_m1), DKK1 (Hs00183740_m1), ZEB1 (Hs00232783_m1), MEN1 (Hs00365720_m1), HAR1B (Hs03299152_m1), and YAP1 (Hs00371735_m1) following the manufacturer’s protocol. Gene expression was quantified using a comparative Ct method and HMBS and B2M were used as housekeeping genes (Hs00609297_m1 and Hs99999907_m1, respectively) as previously described [24 (link)].

Flies were lysed in 300 µL of distilled water. Protein amount was determined by NanoDrop^® ND-1000 UV-Vis (Thermo Fisher scientific, Waltham, MA, USA) and 900 µL chloroform:methanol (2:1, v/v) were added. After centrifugation, the lower phase was isolated according to the Folch protocol [26 (link)] and dried. Twenty microliters of chloroform:methanol (2:1) were added and the samples were separated by Thin Layer Chromatography (TLC; Silica gel 60A; Sigma-Aldrich, St. Louis, MO, USA) in chloroform:butanol:ethyl acetate:0.25% KCl:methanol (25:25:25:9:16, by vol.). The TLC plates were developed with primulin reagent (Sigma-Aldrich, St. Louis, MO, USA) and quantified by ChemiDoc™ XRS (Bio-Rad laboratories, GmbH, Munich).

Mt coelomic concentration was determined by the spectrophotometric method previously described [46 , 47 ] in either the whole organism or the coelomic fluid. Briefly, tissues were homogenized (1 : 3 wt/vol for whole organism or 1 : 3 vol/vol for coelomic fluid) in the following buffer: 0.5 M sucrose, 20 mM Tris-HCl buffer, pH 8.6, added with 0.006 mM leupeptin, 0.5 mM phenylmethylsulphonilfluoride as antiproteolytic agents, and 0.01% β-mercaptoethanol as a reducing agent. Then, the homogenate was treated to obtain a partially purified Mt fraction by ethanol/chloroform precipitation. Mt concentration in the samples was quantified by spectrophotometric titration of the sulphydryl residues using the Ellman's reagent (5,5′-dithiobis-2-nitrobenzoic acid) using reduced glutathione (GSH) as a standard. Data were expressed as μg MT/mg of proteins.
Protein concentration was quantified by the Bradford assay [48 (link)] using NanoDrop ND-1000 UV-Vis (Thermo Scientific, Waltham, MA, U.S.A.).