Cytotoxicity detection plus kit

Cell viability and cytokine release were evaluated in the supernatants. Viability and proliferation were assessed by using the Cytotoxicity Detection Plus kit (Roche, Basel, Switzerland) based on the measurement of lactate dehydrogenase (LDH) activity. LDH test was conducted following the manufacturer’s recommendations. Viability was evaluated as LDH released from the cytosol of damaged cells into the supernatant. To analyze proliferation, cells were incubated with lysis buffer prior to collecting the supernatant.
The release of interleukins IL1β, IL6, IL10, and GM-CSF was quantified from supernatants by ELISA kits (Ebiosciences Santa Clara, CA, USA, for all, except Diaclone; Bensacon Cedex, France, for IL1β), as previously described [28 (link)].

An XTT assay was performed using tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, Thermo Fisher Scientific, Waltham, MA, USA) and phenazine methosulfate (PMS, Thermo Fisher Scientific, Waltham, MA, USA). After the incubation of cells with a mixture of 0.2 mg/mL XTT and 0.015 mg/mL PMS in medium with 0% FCS for 2 h, absorbance at 450 nm was measured.
ATP assay and LDH assays were performed using an ATPlite assay kit (PerkinElmer, Waltham, MA, USA) or Cytotoxicity Detection Plus kit (Roche, Basel, Switzerland), respectively, according to the manufacturers’ instructions. Caspase-3 activation was measured by a Caspase Glo assay kit from Promega (Fitchburg, WI, USA).