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DYSREGULATION OF THE (IMMUNO)PROTEASOME PATHWAY IN MCD
tubers. We also semi-quantitatively evaluated the IR (nucleus and cytoplasm in glial and neuronal cells) of β1, β1i, β5 and β5i. The intensity of the staining was evaluated using a scale of 0-3 (0: no; 1: weak; 2: moderate; 3: strong staining). All areas of the lesion were examined and the score represents the predominant cell staining intensity found in each case. The frequency of β1, β1i, β5 or β5i positive cells [(1) rare; (2) sparse; (3) high] was also evaluated to give information about the relative number of positive cells within the lesion. We also evaluated intensity and frequency of pS6 and IL-1β staining. As described in previous studies 25, 27, the product of the intensity and frequency scores was taken to give the overall score (total score; immunoreactivity score; IRS, Table 3). Quantification of signal intensity using ImageJ software was performed for β1i and β5i subunits (sup- plementary material).
Cell cultures
Primary fetal astrocyte-enriched cell cultures were obtained from human fetal brain tissue (14-19 weeks of gestation) obtained from the HIS-Mouse (human immune sys- tem mouse) facility of the AMC, Amsterdam. All material has been collected from donors from whom a written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic (Heemstede, The Netherlands), these informed consents are kept together with the medical record of the donor by the clinic. Tissue was obtained in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. Cell isolation was performed as described elsewhere 28-30. Briefly, after removal of blood vessels, tissue was mechanically minced into smaller fragments and enzymatically digested by incubat- ing at 37˚C for 30 minutes with 2.5% trypsin (Sigma-Aldrich; St. Louis, Mo, USA). Tissue was washed with incubation medium containing Dulbecco’s modified Eagle’s medium (DMEM)/HAM F10 (1:1) medium (Gibco, Life Technologies, Grand Island, New York, USA), supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin and 10% fetal calf serum (FCS; Gibco, Life Technologies, Grand Island, New York, USA) and triturated by passing through a 70 μm mesh filter. Cell suspension was incubated at 37˚C, 5% CO2 for 48 hours to let glial cells adhere to the culture flask before it was washed with PBS to remove excess of myelin and cell debris. Cultures were subsequently refreshed twice a week. Cultures reached confluence after 2-3 weeks.
Primary FCD astrocyte cultures were derived from a surgical human brain spec- imen obtained from a patient with FCD type IIA (age at surgery: 16 years; female; loca- tion: frontal; seizure frequency: 3/week; duration of epilepsy: 11 years) undergoing epi- lepsy surgery at the Department of Pediatrics / Neurosurgery of the Medical University Vienna (Vienna, Austria). FCD astrocyte cultures were established in the same manner as described above for fetal cultures.
Secondary astrocyte cultures for experimental manipulation were established by trypsinizing confluent cultures and sub-plating onto poly-L-lysine (PLL; 15 μg/ml, Sigma- Aldrich)-precoated 12 and 24-well plates (Costar, Cambridge, MA, USA; 5 ×104 cells/well in a 12-well plate for RNA isolation and PCR; 2.5 X 104 cells/well for immunocytochemistry). In the present study astrocytes were used for analyses at passage 2-4.
Cell cultures were stimulated with human recombinant (r)IL-1 β (Peprotech, Rocky Hill, NJ, USA; 10 ng/ml) or in some experiments with lipo-polysaccharide (LPS; 100
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