Synergy 2 fluorescent plate reader

Caspase assay was performed as per manufacturer's instructions. Briefly, 50000 cells were plated in a 24-well plate and treated with indicated drugs for 4 hours in duplicate. At the end of treatment, cells were centrifuged at 200 rcf for 5 minutes and washed once with 1× PBS and lysed in 50 μL of 1× lysis buffer provided in the kit. For efficient lysis, cells were subjected to a single freeze-thaw cycle. The lysed cells were centrifuged again to remove cell debris and the supernatant was used in the assay. 50 μL of 2× substrate working solution containing Z-DEVD-R110 substrate was added to the cell lysate and incubated at room temperature for 30 minutes. The rhodamine 110-derived substrate (Z-DEVD-R110) used in this assay is a non-fluorescent bisamide compound that, upon enzymatic cleavage via active caspase 3 and may be caspase 7 in the cell lysates, is converted in a two-step process to the fluorescent monoamide and then to the even more fluorescent R110 product. Both of these products are then measured using Biotek synergy 2 fluorescent plate reader at corresponding wavelengths (excitation 496 nm/emission 520 nm). The fluorescent reading was then normalized to the amount of protein in the cell lysate determined via standard BCA assay (Pierce ThermoFisher Scientific).

Cortical neurons were seeded into 96-well plates at 3.5 × 10⁴ cells per well and cultured until 7 DIV at 37°C and 5% CO₂. 20 µM of FITC conjugated CBD3 peptide aptamers were added on neurons and allowed to penetrate the cells for 10 min. After four washes with phenol red free Leibovitz medium, the FITC fluorescence in the cells was measured using a Synergy 2 fluorescent plate reader (Biotek, Winooski, VT, USA) at an excitation wavelength of 485 nm and emission wavelength of 530 nm. Efflux of the peptides from the cells was assessed by measuring FITC fluorescence in the media after 0, 10, 30 and 60 min. To correct for any differences in cell plating, at the end of the experiment cortical neurons were lysed with 20 mM Tris, pH 7.4, 50 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS Protease inhibitor cocktail set III (Calbiochem), phosphatase inhibitor cocktail set I (Calbiochem), 50 U/ml benzonase (Merck) and the protein quantity determined with a Pierce assay.