Opteia mouse il 6 elisa kit

The cortices from B6/C3H P0-P2 mice were isolated as previously described [60 (link)]. The mixed glial cultures were maintained in DMEM/10 % FBS with 100 units/ml penicillin and 100 μg/ml streptomycin. After 10–14 days of incubation, flasks were shaken at 150 rpm for 30 min at 37 °C to dislodge microglia from the astrocyte layer. The microglial cells were then re-plated into 6 well culture dishes in DMEM/10 % FBS with 100 units/ml penicillin and 100 μg/ml streptomycin. All cells were maintained at 37 °C in a humidified incubator with 5 % CO2. When the primary microglial cultures reached ~60 % confluency, the media was removed from the wells and was replaced with 2 ml of media containing 50 ng/ml LPS. Control wells received the same treatment, without the addition of LPS (n = 3/group). The cultures were returned to the incubator for 1, 6 or 12 h. Following incubation, images of the cultures were obtained using an EVOS FL cell imaging system (AMG). Then, the media was removed, mixed with protease inhibitors and aliquoted. The amount of IL-6 within the media (diluted 1:50) was determined using a BD OptEIA mouse IL-6 ELISA kit according to the manufacturer’s protocol (BD biosciences).

After infection for 6, 24 and 48 h, cell culture media were collected for quantitative determination of the lactate dehydrogenase (LDH) activities by LDH Cytotoxicity Assay Kit (Beyotime, Haimen, China) and the proinflammatory cytokines IL-6, TNF-α and IL-1β by employing BD OptEIA™ Mouse IL-6 ELISA Kit (BD), BD OptEIA™ Mouse TNF ELISA Kit (BD) and Mouse IL-1 beta/IL-1F2 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s manual. Total mRNA from infected mice tissues was extracted using the RNeasy Plus Mini kit (QIAGEN, Hilden, Germany), followed by reverse transcription. mRNA levels of proinflammatory cytokines IL-6, TNF-α and IL-1β were determined by RT-PCR using primers listed in Table 1.