Glomax multi elisa reader

Cytotoxicity of tumor cells by CIK cells, which were cocultured with CD40L-expressing DC towards different tumor cells (EgI-1, LoVo, DanG, TFK-1) was determined using the Cytotox-One Homogeneous Membrane Integrity Assay (Promega, Mannheim, Germany), following the manufacturer’s instructions. This assay is based on the release of lactate dehydrogenase (LDH) from damaged cells. Fluorescence signal was measured with a GloMax Multi ELISA reader (Promega, Mannheim, Germany). The different target cells were incubated with the CIK effector cells at different ratios for 4 h. Maximum release was obtained by incubating target cells with lysis buffer. Target cells with effector cells at different ratios served as negative control (spontaneous release). Specific lysis was calculated as follows: Percent cytotoxicity = (experimental release − spontaneous release)/(maximal release − spontaneous release).

One day prior to transfection, 10⁴ cells were seeded onto 96-well-plates and then cultivated in antibiotic-free medium. 100 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cells 48 hours after transfection. Cells were then incubated for 45 minutes. Supernatants were removed and MTT was solubilized with 100 μL dimethyl sulfoxide (DMSO). Optical density was measured colorimetrically at 560 nm (GloMax Multi ELISA reader, Promega, Mannheim, Germany).
To test for effects on tumour cell proliferation, cells were labelled using 5-bromo-2′-deoxyuridine (BrdU) in accordance with the manufacturer's protocol (Cell Proliferation ELISA, Roche Diagnostics, Mannheim, Germany) 24 hours after transfection. Another 24 hours later, cells were fixed and BrdU incorporation was determined using a specific antibody. Optical density was determined at 450 nm (GloMax Multi ELISA reader).
Apoptosis was determined by quantifying cytoplasmic histone-associated DNA fragments. The Cell Death Detection kit (Roche Diagnostics) was applied following the manufacturer's protocol. 48 hours after transfection, cells were lysed and histone-associated DNA was determined using specific antibodies and ELISA technique. Optical density was determined at 405 nm (GloMax Multi ELISA reader).

For analysis of (phosphorylated) STAT3, 10⁴ cells were seeded on a black 96-well tissue culture plate with clear bottom. The cells were transfected as described above. Two days after transfection, STAT3 phosphorylation was quantified with a cell-based STAT3 Immunoassay (R&D Systems, Wiesbaden, Mannheim, Germany) as described in the manufacturer's protocol. STAT3 and phosphorylated STAT3 (pSTAT3) were detected with two different specific antibodies and corresponding fluorescent secondary antibodies.
Fluorescence was measured with the GloMax Multi ELISA reader (Promega). For STAT3, fluorescence was measured at 360 nm (excitation) and 450 nm (extinction) and at 540 nm (excitation) and 600 nm (extinction) for pSTAT3.