5424 microcentrifuge

To determine the mass of mRNA loaded, mRNA loaded in a RNApatch was first separated by scraping the needles into an eppendorf tube and then re-dissolved with RNase-free water. The quantity of mRNA present was then determined using Nanodrop 2000 Spectrophotometer. A blank microneedle patch containing no mRNA was used as control to account for background absorbance caused by PVP.
To study the solubility limits of mRNA in the PVP formulation, known quantities of mRNA were lyophilized, re-constituted in nuclease free water and mixed with PVP (1000 g/L) in a 1:4 (v:v) to achieve desired concentrations of 5, 8, 10 and 20 μg/μl. After mixing, each sample was centrifuged using the Eppendorf 5424 Microcentrifuge at max speed for 1 minute. The supernatants were completely removed and transferred into new tubes for analysis. Gel pellets, if any, were washed by resuspending them with 100 μl nuclease free water, followed by centrifugation again at max speed for 1 minute. Supernatant were discarded and washed pellets, if any, were either directly used for analysis or further treated with 20 mM EDTA. Gel electrophoresis was employed to evaluate presence of mRNA in the supernatant, washed pellets, and EDTA treated washed pellets. The absorbance spectrum was characterized using Nanodrop 2000C Spectrophotometer using the UV-Vis mode.

The amount of extracellular DNA (eDNA) was measured after its precipitation from cell-free supernatants. For this, 500 µL of the aforementioned biofilm culture and 550 µL of the bottom culture containing solid–liquid interface biofilms of the same microcosm, in which the PA-11/PHMG-NS films had been incubated for 72 h, were vortexed for 1 min and spun down at 13,000× g for 15 min (Eppendorf 5424 Microcentrifuge). Five hundred microlitres of each supernatant were removed and transferred into new 1.5 mL plastic tubes. One millilitre of chilled 96% ethanol and 50 µL of a 3 M sodium acetate solution (pH 5.2) were added to the supernatants, which were then incubated overnight at –20 °C. Then, the samples were spun down at 13,000× g for 15 min and the supernatants were removed. One millilitre of 70% ethanol was added into each of the tubes, which were then spun down again at 13,000× g for 15 min to wash the pellet. The supernatants were removed and the sediments were dried to full ethanol evaporation. Two hundred microlitres of Tris-EDTA (TE) buffer were added to dissolve each sample. eDNA concentration was measured using a NanoDrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Total protein was extracted from 1 × 10⁶ cells/T25 of MDA-MB-231 cells after 24 hours of treatment with 3, 10 and 30 μM of Artonin E. Briefly, treated and untreated samples were lysed with 100 μL RIPA lysis buffer containing 1 μL of E-64 protease inhibitor cocktail. The pellet was resuspended thoroughly and incubated at 4°C for 30 minutes with agitation before centrifuging it at 10,000 rpm (Eppendorf 5424 Microcentrifuge, USA) for 10 minutes. The supernatant was thereafter transferred into fresh Eppendorf tubes and the concentration of the extracted proteins was quantified using Pierce BCA protein assay kit by microplate assay (universal microplate reader, Biotech, Inc, USA). This was done in three replicates.