Optiplate 96 f

The activity of β-glucocerebrosidase, β -galactosidase, and β-hexosaminidase at the plasma membrane level was assayed using a high-throughput cell lived-based assay as previously described (Aureli et al. 2011 (link)). Cells were plated in 96-well microplate at a density of 3000 cells/cm²; 24 h after plating, cell culture medium was removed and cells were rinsed with DMEM-F12 without phenol red. Artificial substrates (MUB-Gal, MUG), solubilized in DMEM-F12 without phenol red at pH 6 at a final concentration of 1 mM and 6 mM, respectively, were added to the cells. For β-glucocerebrosidase assay, cells were pre-incubated for 30 min at room temperature with 5 nM AMP-DNM in DMEM/F-12. After 2 h incubation at 37 °C, 10 µl of the reaction mixtures was transferred to a black microplate (Black, 96-well, OptiPlate-96 F, Perkin Elmer) and 190 µl of 0.25 M glycine pH 10.7 was added. The MUB-associated fluorescence was detected by the Victor microplate reader (ex/em 365/460 nm). Since fluorogenic substrates are not able to cross the plasma membrane, under these experimental conditions, the measured fluorescence is exclusively attributable to the hydrolytic activity of PM glycohydrolases. Enzymatic activity was expressed as nanomoles of product/hour/10⁶ cells.

The nonfluorescent Calcein AM is a cell-permeant dye that in live cells is converted to a green fluorescent calcein after hydrolysis by intracellular esterases. Cells were plated in 96-well microplate at a density of 3000 cells/cm² and loaded or not for 30 days with SM. After culture medium removal, cells were washed with PBS and then incubated with 5 µg/ml Calcein AM solution in PBS (15 min, 37 °C). At the end of incubation, Calcein-AM solution was discarded and cells were lysed with 1% Triton X-100 in TNEV buffer (10 mM TrisHCl pH 10, 150 mM NaCl, 5 mM EDTA pH 7.5) in mild agitation (10 min, 23 °C). The cell lysates were transferred to a black microplate (Black, 96-well, OptiPlate-96 F, Perkin Elmer) and fluorescence intensity (ex/em 495/515 nm) was measured by the Victor microplate reader.

In order to explore the effect of let-7 miRNA on the level of oxidative stress, we carried out estimation of ROS levels using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay following the protocol as described by Kaur et al. (2012) (link). Worms of control and let-7 miRNA silenced groups were washed thrice with M9 buffer and twice with phosphate buffer saline (PBS). An approximate number of 100 worms/100 μl assay solution, were transferred to assay wells of OptiPlate-96 F (Perkin Elmer) with each group being assayed in triplicates. A volume of 100 μl H₂DCFDA (Cat. No. D399, Invitrogen) from an ethanol stock of 100 μM, was added to each well. Fluorescence from each well was quantified at three time points – (i) before addition of the dye, (ii) immediately after addition of the dye, and (iii) post 1 h incubation of addition of the dye. The Fluorescence intensity measurements were carried out using Multimode plate reader (Perkin Elmer, VICTOR^TM X3), at excitation wavelength 485 nm and an emission wavelength 520 nm. The change in fluorescence was calculated by subtracting initial reading from the final reading; the numbers were presented as fluorescence intensity per worm and plotted as mean ± SE. Statistical significance was calculated by student’s t-test using GraphPad Prism software package.