Twenty-four hours prior to the measurements, the cells in culture flasks were provided with fresh DMEM medium (37 °C, pH 7.1) supplemented with 100 mM or 200 mM trehalose (Sigma). Consequently, the cells were thoroughly washed (4 × 1 min) in fresh DMEM without trehalose (as proved by LC-MS/MS), harvested by scratching, resuspended in 1 mL of DMEM (1xPBS for DESI), disintegrated by shaking with glass bead tubes 0.5 mm (MO BIO Laboratories, Carlsbad, CA), with microtube homogenizer (BeadBug microtube homogenizer D 1030-E, Benchmark Scientific, Edison, NJ), 3 min, 4000 rpm, all extracts were centrifuged (6000 rpm/15 min, HERMLE Z326K, Germany) to remove cells debris and glass pellets, and 100× diluted for the measurements. Filtered (Nylon filter Mini-UniPrep 0.45 μm pore size, Whatman, UK) cell supernatants were used for DART and LC-MS/MS. For DESI-MS the cells were cultivated the same way as for DART. Cell culture was rinsed and centrifuged. The pellet was loaded onto a nylon membrane Nylon 66, 0.2 μm (Supelco, Bellefonte, PA). 2 μL of cell suspense were loaded using the MICROMAN pipette (Gilson, Villiers-le-Bel, France). Nylon membrane was fixed to the glass slides (Prosolia, Indianapolis, IN) by the means of double-site tape.
Beadbug microtube homogenizer d 1030 e
Manufactured by Benchmark Scientific
Sourced in United States
The BeadBug microtube homogenizer D 1030-E is a compact and versatile laboratory instrument designed for efficient homogenization of small samples. It utilizes rapid bead-beating action to disrupt cells, tissues, and other materials within microtubes, facilitating effective sample preparation for various analytical and research applications.
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2 protocols using beadbug microtube homogenizer d 1030 e
1
Trehalose Supplemented Cell Preparation Protocol
2
Asiatic Pennywort Extract Preparation
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Leaves incubated for 1, 2, and 3 days were dried in a hot air oven at 60 °C for 48 h. The C. asiatica extract was carried out according to a modified method of Alqahtani et al. (2015) [11 (link)]. The dried leaves were then weighed at 100 mg, homogenized to powder using a BeadBug Microtube Homogenizer D1030-E (Benchmark Scientific, USA), and passed through a 2 mL sieve. The C. asiatica extract was obtained by adding 1 mL of methanol to the powder and mixing in a vortex mixer at 3500 rpm for 15 min before sonication in an ultrasonic bath for 15 min and centrifugation at 3500 rpm for 5 min. The supernatant was removed, and the rest of the pellet was repeated for extraction once. The supernatant collected from two rounds was pooled and filtrated through a 0.45 μm nylon membrane, with the volume adjusted to 2 mL by adding methanol (HPLC-grade, RCI Labscan LIMITED, Thailand) and storing at −20 °C for further chemical analysis.
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