Evaluation of Microglial Cytotoxicity

The kit was utilised in order to analyse the effect of microglia conditioned medium on neuronal cytotoxicity. CATH.a cells were seeded in 96-well plates and allowed to adhere. Following overnight serum starvation, the cells were incubated in the presence of microglia-conditioned medium (microglia cells were treated with LPS in the absence or presence of the autotaxin inhibitor PF8380). Three wells containing only medium without cells were used as background control. As a positive control, cells were incubated with the LDH positive control solution (100% release). In order to measure maximum and spontaneous release, cells were incubated with 10% Triton X-100 and assay buffer, respectively.

LPS increased NO production (detected as nitrate), and ATX and inhibition reduced nitrate levels. To determine potential neurotoxic properties of the LPS-induced secretome of BV-2 cells, murine CATH.a neurons were incubated with supernatants that were collected from LPS- or LPS+ inhibitor-treated BV-2 cells. These experiments revealed significant neurotoxicity of the media that were collected from LPS-activated cells. In contrast, the pre-conditioned medium that was obtained from LPS+ inhibitor-treated cells did not affect neuronal viability, and induced a slightly lower lactate dehydrogenase (LDH) release from the neuronal cultures as compared to the medium that was collected from the untreated cells.

The assay is simple and the kit easy and fast to use, providing us with reproducible and constant results between experiments.