RNA was extracted directly from agar plugs. Frozen assay blocks were thawed in a 37°C water bath and spun down at 2000 rpm for 2 min in a Heraeus Megafuge 16 tabletop centrifuge equipped with Thermo M-20 swinging bucket plate rotor. The Zymo Quick-RNA Viral Kit extraction protocol was followed using 96-well plates. Filter and collection plates were provided with the kit. No DNA/RNA Shield was used. Samples were eluted in 40 μL nuclease-free water into MicroAmp Optical 96-well reaction plates (Applied Biosystems). RNA can be covered and stored at −80°C or used directly for reverse transcription.
Megafuge 16
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Megafuge 16 is a high-performance floor-standing centrifuge designed for a wide range of laboratory applications. It features a compact design and can accommodate various rotor options to handle different sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Dispase solution, by Roche (2 mentions)
Microamp optical 96 well reaction plate, by Thermo Fisher Scientific (1 mentions)
Quick rna viral kit, by Zymo Research (1 mentions)
Anhydrous na2so4, by Merck Group (1 mentions)
Rotavapor r 215, by Büchi (1 mentions)
Multi reax, by Heidolph (1 mentions)
Microfuge r, by Beckman Coulter (1 mentions)
100 m nylon mesh, by BD (1 mentions)
6 protocols using megafuge 16
1
Rapid Viral RNA Extraction from Agar Plugs
2
Encapsulation of Dye-Loaded Polystyrene Microparticles
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1 μm sized aminated polystyrene microparticles (PSMPs) were bought from Micromod GmbH (Rostock, Germany). The highly hydrophobic complex was encapsulated in the 1 μm sized aminated polystyrene microparticles (PSMPs) via a simple one-step staining procedure as follows: First, 50 mg/mL of PS microparticles were diluted in deionized water to 2.5 mg/mL (1.2 mL). A 0.8 mM solution of the ZnNMe2Nc complex in tetrahydrofuran (THF) was prepared. Then, 200 μL of this solution in THF was added to the PSMP dispersion in water, and the sample was subsequently shaken for 40 min. Shrinkage of the particles was induced by adding 200 μL of deionized water to the dispersion, thereby encapsulating the complex in the microparticles. The particles were then centrifuged at 16.000 rpm for 5 min (Megafuge™ 16 from Thermo Scientific, Schwerte, Germany). The precipitate was washed three times with ethanol/water mixtures (volume ratios of 40/60, 30/70, and 20/80) and once with deionized water to remove the excess of dye that was adsorbed onto the particles’ surface. Finally, the suspension containing the PSMPs loaded with the ZnNMe2Nc complex was diluted to 5 mg/mL (600 μL).
3
Microbial Lipid Extraction and Quantification
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Total lipid extraction was conducted using the method described by Folch [8 ] with some modifications [34 (link)]. Briefly, cell biomass (0.4 g/L) was centrifuged at 5000 rpm for 10 min at 4 ºC (Heraeus, Megafuge16, Thermo Scientific) and dried at 105 ºC overnight. Dry biomass (around 0.2 g dry weight) was mixed with a solution of chloroform and methanol (2:1 v/v) under reflux for 4 h. The extract was filtered (Watman N.1 paper) and 0.88% KCl solution was used to wash the organic phase. Samples were dried using anhydrous Na2SO4 (Sigma). The chloroform phase containing the lipids was evaporated using a Rotavapor R-215 (BUCHI) at 40 ºC and 350 mb of vacuum. Finally, total cellular lipids were gravimetrically determined and expressed as grams of lipid per grams of dry biomass (% w/w).
A parametric one-way ANOVA and F-test were used for the assessment of means and variances of microbial lipid content (% w/w) (confidence interval 90%), respectively. Differences were considered significant at p-value < 0.05.
A parametric one-way ANOVA and F-test were used for the assessment of means and variances of microbial lipid content (% w/w) (confidence interval 90%), respectively. Differences were considered significant at p-value < 0.05.
4
Isotope Dilution and Matrix-Matched Calibration for Accurate Mycotoxin Quantification
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To improve the accuracy of the measurements, for the analytes for which U-[13C]-labelled homologues were available, standard isotope dilution assay (SIDA) was performed. In the case of the sulfates derivates, matrix-matched calibration was carried out. For that, several blank oat samples were extracted by weighting 5 g of oat flour and mixing it with 10 mL acetonitrile and 10 mL water, shaking for 30 min (Multi Reax, Heidolph Instruments GmbH and Co.KG, Schwabach), centrifuging at 3800 g for 30 min (Megafuge 16, Thermo Fisher Scientific, Braunschweig, Germany), collecting 1 mL of the supernatant and mixing it with 250 mg of anhydrous magnesium sulfate. Then, the samples were centrifuged at 17,000 g for 5 min (Microfuge R, Beckmann, Krefeld, Germany) and all the resulting extract collected in a vial for further use.
Finally, three eight-point series of calibration solutions were prepared: one containing ZEN (8.008–500.5 ng/mL), α-ZEL (1.1047–69.074 ng/mL), β-ZEL (2.776–173.5 ng/L) and their respective U-[13C]-labelled homologues, dissolved in acetonitrile:water (25:75, v/v); one containing a series of calibration solutions of ZEN-14-S (0.858–546.25 ng/mL) and another one containing α-ZEL-14-S (0.853–947.1 ng/mL) and β-ZEL-14-S (0.6092–676.35 ng/mL), both dissolved in blank matrix extract (25%).
Finally, three eight-point series of calibration solutions were prepared: one containing ZEN (8.008–500.5 ng/mL), α-ZEL (1.1047–69.074 ng/mL), β-ZEL (2.776–173.5 ng/L) and their respective U-[13C]-labelled homologues, dissolved in acetonitrile:water (25:75, v/v); one containing a series of calibration solutions of ZEN-14-S (0.858–546.25 ng/mL) and another one containing α-ZEL-14-S (0.853–947.1 ng/mL) and β-ZEL-14-S (0.6092–676.35 ng/mL), both dissolved in blank matrix extract (25%).
5
Isolation of Human Primary Skin Fibroblasts
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Human primary skin fibroblasts were isolated as described previously (Szebeni et al., 2018 (link)). Briefly, healthy volunteers (age 18–60 years) were enrolled into the study. The punch biopsies were taken from healthy subjects from the breast area undergoing plastic surgery. Primary fibroblasts were obtained from the skin by enzymatic digestion according to a standard protocol. Briefly, skin specimens were first washed in Salsol A solution (Human Rt) supplemented with 2% antibiotic/antimycotic solution (Sigma‐Aldrich). Skin samples were then cut into narrow strips and incubated in Dispase solution (Roche Diagnostics, Mannheim, Germany) overnight at 4°C. The epidermis was subsequently separated from the dermis. Fibroblasts were obtained by incubating the dermis in Digestion Mix solution (Sigma‐Aldrich, Collagenase, Hyaluronidase, and Deoxyribonuclease) at 37°C for 2 h. Cell suspensions were filtered through a 100 µm nylon mesh (BD Falcon), and cells were pelleted by centrifugation (Megafuge 16; Thermo Fisher Scientific). Fibroblasts were grown in low glucose DMEM/F12 medium containing 15% FCS, 1% antibiotic/antimycotic (PAA, Pasching, Austria) and 1% l ‐glutamine solution (PAA). Fibroblasts were cultured at 37°C and 5% CO2 in humidified conditions. Depending on the cell growth, the medium was changed every 2–4 days, and cells were passaged at 80% of confluence.
6
Primary Fibroblast Isolation from Skin Biopsies
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Healthy volunteers (age 18–60 years) were enrolled into the study. The punch biopsies were taken from healthy subjects from the breast area undergoing plastic surgery. Primary fibroblasts were obtained from the skin by enzymatic digestion according to a standard protocol. Briefly, skin specimens were first washed in Salsol A solution (Human Rt, Gödöllő, Hungary) supplemented with 2% antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA). Skin samples were then cut into narrow strips and incubated in Dispase solution (Roche Diagnostics, Mannheim, Germany) overnight at 4 °C. The epidermis was subsequently separated from the dermis. Fibroblasts were obtained by incubating the dermis in Digestion Mix solution (Collagenase, Hyaluronidase and Deoxyribonuclease) for 2 h at 37 °C. Cell suspensions were filtered through a 100 µm nylon mesh (BD Falcon, San Jose, CA, USA), and cells were pelleted by centrifugation (Thermo Fisher Scientific, Waltham, MA, USA, Megafuge 16). Fibroblasts were grown in low glucose DMEM/F12 medium containing 15% FCS, 1% antibiotic/antimycotic (PAA, Pasching, Austria) and 1% l -glutamine solution (PAA). Fibroblasts were cultured at 37 °C and 5% CO2 in humidified conditions. Depending on the cell growth, the medium was changed every 2–4 days, and cells were passaged at 80% of confluence.
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