Ldh based cytotoxicity detection kit

In order to evaluate vitality of cells in our different culture setups, lactate dehydrogenase (LDH) concentration in supernatants of BMSC, mixed cultures and chondrocytes was determined in monocultures and co- or tricultures with OAB kept in chondrogenic medium. Content of LDH was analyzed at days 7, 14, 21 and 28 with an LDH-based cytotoxicity detection kit (Roche Diagnostics, Penzberg, Germany) and compared to respective assay controls (high control = all cells in fibrin gels were lysed; low control = spontaneous cell death of an equivalent cell amount in monolayer) according to the manufacturer’s instructions. LDH concentration released from dead cells into supernatant was determined on a photometrical basis at absorption of 490 nm (Tecan GENios with Magellan 6.5; Tecan, Crailsheim, Germany). Due to high interexperimental variability - presumably caused by personal living conditions and physical activity, medical treatment or general health status of tissue donors - we have calculated the raw data as percentage of control per individual experiment. Repetition was in triplicate at least three times with cells and explants from different donors.

The effect of DMF on cell proliferation was measured by quantifying BrdU via a cell proliferation immunoassay from Roche Diagnostics (Grenzach, Germany). Forty-eight hours after seeding (1 × 10⁴ cells per 96-well), cells were incubated with BrdU and DMF at the indicated concentrations for 24 h. The cytotoxic potential of DMF was determined using an LDH-based cytotoxicity detection kit from Roche. Forty-eight hours after seeding (1 × 10⁴ cells per 96-well), the cells were incubated under serum-reduced conditions (1% FCS) with DMF for 24 h at the indicated concentrations.