Viacount kit

Mononuclear cells were isolated from mice spleens by Ficoll-Hypaque density gradient centrifugation and the count and viability of the lymphocytes were determined using a Guava EasyCyte Flow Cytometer and Viacount Kit (both Merck, USA). For this, 2 × 10⁶ cells/ml were placed in RPMI containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin, and with/without 10 μg/ml of recombinant gB. After incubation for 20 h at 37°C, the cells were used for phenotypic analysis of lymphocytes. Then, the cells were washed three times with PBS and viability staining was performed with FVS780 dye (Becton Dickinson, USA) according to the manufacturer’s protocol. Subsequently, the cells were washed and placed in vials containing PBS supplemented with 2% FBS and 0.09% sodium azide (staining buffer). Cell surface marker staining was then performed on the combination with the following conjugated antibodies: CD3-FITC, CD4-PE, CD8-BV650, and CD69-BV421 (all from Becton Dickinson). After incubation in the dark for 30 min on ice, the cells were washed three times with staining buffer. Subsequently, at least 10.000 CD3⁺ events were analyzed by flow cytometric acquisition using the Guava EasyCyte 16ST Flow Cytometer (Merck). Data were analyzed using InCyte software (Merck).

A culture medium with or without phenol red (RPMI 1640); dihydroethidium (DHE); Fura-2 AM and proinflammatory cytokines (IL-1β, TNF and IFN-γ) from Thermo Fisher Scientific (Waltham, MA, USA); collagenase P and 4-phenylbutyric acid (4-PBA) from Sigma-Aldrich (St. Louis, MO, USA); GSK2795039 from Hycultec (Beutelsbach, Germany); a FITC Annexin V Apoptosis Detection Kit with PI from BioLegend (San Diego, CA, USA); a ViaCount kit from Merck Millipore (Burlington, MA, USA); an Insulin Ultra-Sensitive Assay kit from Cisbio (Codolet, France) and anti-p-eIF2-α (#9721S), anti-p-IRE1 and anti-α-tubulin (#3873) antibodies from Cell Signaling Technology (Danvers, MA, USA).