Chapter 5 172 minute 98°C, followed by 30 cycles of 98°C for 5 seconds, 60°C for 15 seconds, 72°C for 15 seconds. After the cycles the PCR was and finalized with 30 seconds of 72°C. Products were loaded onto 1.5% agarose gel and ran for 40 minutes at 125V. Neuron-microglia co-culture Co-cultures were started by thawing and plating day 18 neurons and primary derived rat astrocytes onto PLO / mLam coated plates in NB+ medium supplemented with BDNF, GDNF, IGF, cAMP and 10 µM Y-27632. Rat astrocytes were obtained by papaïn dissociation of cortex of postnatal day 1 pups. To minimize contamination of other cells rat astrocytes were vigorously tapped before each medium change. Astrocytes of passage 3 were used for experiments. For multi-electrode array (MEA) cultures 120k neurons were plated together with 20k rat astrocytes on 24 well MEA plates (Multi Channel Systems, 24W300/30G-288). For cellomics purposes 12k neurons were plated together with 2k rat astrocytes on the CELLSTAR® 96 wp µClear®. For the ALD and 4H microglia-like cell co-cultures, a mixture of neurons from line hvs088, hvs420, hvs451a and H01 in equal parts was made. Half of the medium was replaced biweekly, with supplements for the entire volume, Rock-Inhibitor Y27632 was only supplemented at plating. Co-cultures were started at day 53 neurons, at the beginning of the 6th week. From this point the culture were divided in two groups; with and without microglia-like cells. The without microglia group received the same media changes as the with microglia-like cells group, but microglia-like cells were not plated. The microglia containing group got 3k microglia-like cells in each well of the 96wp and 30k in the 24w MEA plates. The plating medium was coculture medium consisting of 50% NeurobasalTM and 50% DMEM/F-12 (Gibco, 21331020) with 1x N1 (Sigma-Aldrich, N6530), 1x B27 (without Vitamin A, Gibco, 12587001), 60 ng/ml T3 (Sigma-Aldrich, T6397), 100 ng/ml Biotin (Sigma-Aldrich, B4639), 1x GlutaMAXTM and 1x P/S supplemented with 20 ng/ml BDNF, 1 µM cyclic-AMP, 100 ng/ml IL-34 (PeproTech, 200-34) and 10 ng/ml GM-CSF (Gibco, PHC2015). At plating also 10 µM of Rock-Inhibitor Y-27632 was added to the medium. Half of the medium was replaced biweekly, with supplements for the entire volume. MEA Baseline activity of neuronal networks was measured according to a previously described procedure (Dooves et al., 2023). Briefly, activity was recorded for 10 minutes at a sampling rate of 10 kHz and at 37°C in controlled atmosphere (humidified air with 5% CO2) using the
RkJQdWJsaXNoZXIy MTk4NDMw