Cytotox one reagent

To evaluate the cytotoxicity, a lactate dehydrogenase (LDH) release assay (Cyto-Tox One Reagent, Promega, Madison, WI, USA) was performed. Primary neuron-astroglia mixed culture or astroglia cells were cultured as described above and then treated with undigested or digested recombinant GFAP protein with either calpain or caspase-6. Culture media were collected 24 h after treatment and assayed for LDH release by following the manufacturer’s instructions. Briefly, 50 μL of the reconstituted 2X LDH assay buffer was added to the 50 μL supernatant. Incubate the plate was incubated at room temperature (22–25 °C) for 10–30 min. Then 50 μL of stop solution was added to the wells. The absorbance was measured at 490 nm. Three to four replicates were assayed.

For the direct assay, 5 mm × 2 mm discs were used. Cells were directly seeded on the discs. Cell proliferation was assessed by absorbance values and the percentage of viable cells was calculated from the obtained values. Cell Titer 96^® Aqueous Assay System, Promega was used for measuring the cell viability and proliferation. MC3T3-E1 (8000 cells) were seeded directly on the discs in 96 well plate. A standard protocol for the MTS assay was followed according to the manufactures recommendations. Optical density values were measured in 96 well plate reader at 490 nm wavelength. (P < 0.05; n = 4). The LDH activity was used as an index of cytotoxicity. CytoTox-One Reagent (Promega) was used for LDH assay. The experiments were done according to the manufacture protocol and the fluorescence values were recorded with excitation of 560 nm and an emission wavelength of 590 nm. (P < 0.05; n = 4). Live-dead cell staining was done using propidium iodide (PI) (Dead cells) stock solution, fluorescein diacetate, and Hoechst (Blue) and examined using an upright fluorescence microscope (Leica DMRB, Leitz). Cell attachment and cytoskeleton morphology were observed using scanning electron microscopy.

All cell viability studies were performed in the presence of FCS. Cells were seeded at 2.5–5 × 10³ cells/well, depending on cell line, in 96-well plates (PAP assays: CHO-K1 at 3 × 10³ cells/well; peptide cytotoxicity assays: CHO-K1 at 5 × 10³ cells/well; Bouganin assay: CHO-K1 and CHO-K1/EGFR at 2.5 × 10³ cells/well; ^DPMIα assays: T47D at 5 × 10³ cells/well; Omomyc assays: all cell lines at 5 × 10³ cells/well). In brief, adherent cells were allowed to adhere for 24 h prior to the addition of treatments whereas suspension cell lines were treated immediately following seeding. Following 2–48 h incubations with treatments, cell viability was measure by a variety of methods. Membrane integrity was assessed by the release of LDH into the media via the CytoTox-ONE reagent (Promega). Metabolic activity was measured either by resazurin reduction potential using PrestoBlue (Life Technologies) or by ATP activity using CellTitre-Glo (Promega). All assays followed manufacturer’s instructions. IC₅₀ values were calculated using Prism (version 7.0a, GraphPad).