Previously, cell-free media obtained from LPS/IFN-γ-exposed microglia-like cells resulted in the highest toxicity on cell viability of SH-SY5Y cells [28 (link)]. To prepare conditioned medium (CM) with inflammatory factors, BV-2 cells were stimulated with a combination of LPS (1 μg/ml) and IFN-γ (100 ng/ml) for 24 h. After morphology examination, the BV-2 CM were collected, pooled, and centrifuged to remove cell debris. The induced inflammation was confirmed by release of NO, TNF-α, IL-1β, and IL-6 in the media and increased Iba1 expression in the cell lysate.
For SH-SY5Y cell viability assay, DMEM-F12 was then mixed with two times volume of BV-2 CM (a final FBS concentration at 10%) and added to undifferentiated ΔK280 tauRD-DsRed SH-SY5Y cells for 2 days to induce inflammation. Cell viability was determined by MTT assay as described. For SH-SY5Y cytotoxicity assay, neuronal-differentiated ∆K280 tauRD-DsRed SH-SY5Y cells were treated with BV-2 CM for 5 days as described and media were collected. 100 μl of supernatant from each sample was transferred to 96-well plate to examine the release of lactate dehydrogenase (LDH) by using LDH cytotoxicity assay kit (Cayman, Ann Arbor, MI, USA). The absorbance was read at 490 nm with a microplate reader (Multiskan GO, Thermo Scientific).
For SH-SY5Y cell viability assay, DMEM-F12 was then mixed with two times volume of BV-2 CM (a final FBS concentration at 10%) and added to undifferentiated ΔK280 tauRD-DsRed SH-SY5Y cells for 2 days to induce inflammation. Cell viability was determined by MTT assay as described. For SH-SY5Y cytotoxicity assay, neuronal-differentiated ∆K280 tauRD-DsRed SH-SY5Y cells were treated with BV-2 CM for 5 days as described and media were collected. 100 μl of supernatant from each sample was transferred to 96-well plate to examine the release of lactate dehydrogenase (LDH) by using LDH cytotoxicity assay kit (Cayman, Ann Arbor, MI, USA). The absorbance was read at 490 nm with a microplate reader (Multiskan GO, Thermo Scientific).