Bioluminescent assay kit

Myc-CaP cells were transfected with siEPHB4 or siCNT, or treated with EphB4 inhibitor for 72 h. After the indicated times, supernatants were collected and cell counts performed for quantifying secreted ATP (Bioluminescent Assay Kit, Sigma) and high-mobility group protein B1 (HMGB1; Elisa, Techan Trading). For detection of surface Calreticulin, cells were incubated with rabbit anti-Calreticulin (1:1000, Abcam ab2907) for 60 min and then incubated with Alexa Fluor 488 anti-rabbit secondary antibody (Invitrogen A11008 1 µg/ml) and analyzed by flow cytometry.

Cell death from CP1 or MG1655 (MOI 1 or 10) and cancer cell co-culture was measured by supernatant lactate dehydrogenase (LDH; Cytotoxic 96 Non-Radioactive Cytotoxicity Assay, Promega). For in vitro ICD assays, 1 μM mitoxantrone was used as a positive control. Supernatants were collected and cell counts performed after 72 h for quantifying secreted ATP (Bioluminescent Assay Kit, Sigma) and high mobility group protein B1 (HMGB1; ELISA, Tecan Trading). Also after 24 or 72 h, cells were incubated with rabbit anti-calreticulin (Abcam ab2907 1:1000) for 60 min, followed by Alexa Fluor 488 anti-rabbit secondary (Invitrogen A11008 1 μg/ml) for 30 min, and analyzed by flow cytometry. For in vivo ICD assays, HMGB1 immunofluorescence (IF) was quantified as the percentage of HMGB1⁻ nuclei and calreticulin IF was analyzed for cell surface staining. In vitro, caspase 3/7 activity was assessed at 6 or 24 h (Caspase-Glo 3/7 Assay, Promega). Early (Annexin V⁺ PI⁻) and late (Annexin V⁺ PI⁺) stage apoptosis were analyzed at 24 h by flow cytometry (Annexin V Apoptosis Detection Kit, eBioscience). Phosphorylated MLKL, MLKL, RIP1, and full length and cleaved PARP were analyzed by Western blot. As indicated, select experiments included the addition of 50 μg/ml gentamicin 2 h after co-culture initiation.