Dimethylsulfoxyde

A suspension of sodium hydroxide in dimethylsulfoxyde was prepared by grinding about 0.90 g of dried sodium hydroxide (4 tablets) in 3 mL of dimethylsulfoxyde (Sigma-Aldrich). 0.6 mL of this preparation was added to the lyophilized sample. Then, 0.5 mL of iodomethane (ACS reagent grade, Acros Organics) was added and the sample was incubated under agitation for 1 h. One mL of water was added to quench the reaction. One mL of chloroform and 3 mL of water (LC–MS grade, Thermo Scientific) were then added to the samples and mixed. When two phases appeared clearly, the upper aqueous phase was discarded, and 3 mL of water were added. This step was repeated until pH was neutralized. The chloroform phase containing permethylated glycans was then dried.

Dimethyl Sulfoxyde (DMSO-Ref D2438) and Thapsigargin (Tg-Ref: T9033) were purchased from Sigma-Aldrich. Fura-2 QBT™ Calcium Kit was purchased from Molecular Devices (Ref: R8198).

For in vitro experiments, cell lines were treated with 10μM Erlotinib (AlfaAesar, Ward Hill, Massachusetts, USA), 30μg/mL Cisplatin (Mylan, Saint-Priest, France) or 0.2mL DiMethylSulfOxyde (Sigma Aldrich, Saint-Louis, Missouri, USA) for 18 hours. In order to demonstrate the specificity of the apoptotic signal using Nucview, cell lines were separated in two wells, in which were added ten μM Ac-DEVD-CHO (caspase 3 inhibitor) or DMSO for an additional 15 minutes. Cells were then harvested and a first sequence of images was acquired using the CellVizio^® system, by direct application of the optical miniprobe (Alveo-Flex AF2040, Mauna Kea Technologies) onto the cell pellets. Cells were re-suspended in 500μL of culture medium containing Erlotinib (10μM), Cisplatin (30μg/mL) or DMSO (0.2mL), and Ac-DEVD-CHO (10μM, Biotium) or DMSO. Ten minutes after addition of C3-NucView (0.2mM, Biotium), a second sequence of images was acquired using the same technique.
For flow cytometry experiments, cells were prepared and treated with Erlotinib (10μM), Cisplatin (30μg/mL) or DMSO (0.2mL), and Ac-DEVD-CHO (10μM, Biotium) or DMSO as described above. C3-NucView was added at 0.2mM. Cells were then analyzed on Cytomics FC 500 (Beckman Coulter, Fullerton, California, USA) within the hour. Five separate sets of experiments were performed.