Biospectrometer basic

The AOAC pH-differential method describes a photometric method for the determination of total monomeric anthocyanin content in fruit juices, natural colorants and wines within the range of 20–3000 mg/L. The results are presented as cyanidin-3-glucoside equivalents. Total monomeric anthocyanin content is determined by using the absorptivity and molecular weight of cyanidin-3-glucoside, 26,900 ${\mathrm{L}·\mathrm{mol}}^{-1}{·\mathrm{cm}}^{-1}$ . Due to the usage of 26,900 ${\mathrm{L}·\mathrm{mol}}^{-1}{·\mathrm{cm}}^{-1}$ as molar extinction coefficient, TAC values are underestimated compared to HPLC reference analysis. Furthermore, true molar is hard to obtain due to the high hygroscopicity of anthocyanins. It is extremely hard to obtain pure crystalline anthocyanin in adequate quantities in pure crystalline form.
For pH-differential method [8 (link)] an Eppendorf BioSpectrometer basic (Hamburg, Germany) was used. Buffer A (0.1-M HCl in H₂O, pH adjusted with HCl to 1.0) and Buffer B (0.4-M NaOAc in H₂O, pH adjusted with NaOAc to 4.5) were used. The samples were diluted at a ratio of 1:30 in buffer A and in buffer B, respectively. Absorption (A) was measured at 520 and 700 nm. TAC was determined by Formula 1. MW stands for the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF represents the dilution factor and ε the molar extinction coefficient (26,900 $\mathrm{L}·{\mathrm{mol}}^{-1} ·{\mathrm{cm}}^{-1}$ ).
$TAC=\frac{A·MW·DF·{10}^3}{\epsilon ·1}$

Cultures of wild type and derived mutants of M. smegmatis were inoculated into 125 mL conical flasks containing 30 mL LBT medium (initial OD₆₀₀ of 0.001), in six biological replicates. Growth was monitored by measuring optical density at 600 nm (1 cm cuvettes; Eppendorf BioSpectrometer Basic); when OD₆₀₀ was above 0.5, cultures were diluted ten-fold in LBT before measurement. Specific growth rate during mid-exponential growth was calculated for each replicate using GraphPad Prism (non-linear regression, exponential growth equation, least squares fit). The long-term survival of the cultures was determined by counting colony forming units (CFU mL^–1) of cultures 21 days post-OD_max. Cultures were serially diluted in HdB containing no carbon source and spotted on to agar plates in technical quadruplicates. After incubation at 37°C for 3 days, the resultant colonies were counted.

At the end of the experiment, healthy and mature leaves were collected from each sapling, and chlorophyll a and b (Chl a and b), and carotenoid content (Cc) were determined following the method of Arnon [40 (link)]. Absorption was measured at 470, 645, and 663 nm using a spectrophotometer (Eppendorf BioSpectrometer ^® Basic; Hamburg, Germany). Proline content (Pc) in leaves was quantified using the ninhydrin method of Bates [41 (link)]. The reaction mixture was extracted with 5 mL toluene, cooled at room temperature and absorbance was measured at 520 nm using a spectrophotometer (Eppendorf BioSpectrometer ^® Basic; Hamburg, Germany) and total phenolic contents (TPC) were determined using the Folin–Ciocalteu reagent method of Ainsworth and Gillespie [42 (link)]. Total soluble proteins (SP) were determined by following the method of Bradford [43 (link)] and total soluble sugars (TSS) were determined using the Anthrone method of Yemm and Willis [44 (link)].

For qPCR analysis of olfactory receptor genes and olfactory markers, OE was dissected from 16 homozygous NPC1^-/- mutants and 5 control wild type mice (NPC1^+/+) of both sexes aged to 8 weeks and treated as described in “Pharmacologic Treatment.” Mice were deeply anesthetized with pentobarbital (90 mg/kg) and then decapitated. The dissected tissues were frozen in liquid nitrogen and stored at -80°C. TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States) was used for homogenization of the tissue, followed by RNA extraction according to the manufacturer’s protocol. After precipitation and drying, RNA was resuspended in an aliquot of RNase and DNase-free water quantified by A_260nm spectrophotometry (BioSpectrometer^® basic, Eppendorf, Hamburg, Germany) and stored at -80°C. cDNA was synthesized with 5 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. Control reactions were performed without MultiScribe Reverse Transcriptase. cDNA was stored at -20°C. The quality of amplified cDNA was controlled using β-Actin PCR.

The protein concentration of samples was determined using a BCA Assay. A PierceTM BCA Protein Assay Kit (#23225) was used for this method. Two different dilutions of the depleted serum samples (1:10 and 1:20) were measured with the assay, which was performed in duplicates. Further, a BioSpectrometer^® Basic (Eppendorf, Hamburg Germany) was used as photometer, and standard curve and protein concentrations were calculated by the instrument software.

For RNA extraction and cDNA synthesis, spleens from 9 homozygous Npc1^−/− and 3 Npc1^+/+ control mice of both sexes were dissected at P65. Mice were deeply anesthetized with pentobarbital (90 mg/kg) and then decapitated. The tissues were collected, flash-frozen in liquid nitrogen and stored at − 80 °C. RNA extraction and cDNA synthesis were performed according to Coiro et al. [40 (link)], with slight modifications. TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for homogenization of the tissue, followed by RNA extraction according to the manufacturer’s protocol. After precipitation and drying, RNA was resuspended in an aliquot of RNase and DNase-free water quantified by A_260nm spectrophotometry (BioSpectrometer basic, Eppendorf, Hamburg, Germany) and stored at − 80 °C. cDNA was synthesized with 5 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Control reactions were performed without MultiScribe Reverse Transcriptase. cDNA was stored at − 20 °C. The quality of amplified cDNA was controlled using β-Actin PCR.