75 cm2 cell culture flasks

Human Caucasian breast adenocarcinoma cell line, MDA-MB-231 (ECACC 92020424), was purchased from ECACC (European Collection of Authenticated Cell Cultures), whereas MCF-7 (ATCC HTB-22) was purchased from ATCC (American Type Culture Collection). MCF-7 and MDA-MB-231 were cultured in high-glucose DMEM (Lonza, Basel Switzerland) supplemented with 10% fetal bovine serum (EuroClone), 2 mM l-glutamine, and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). MCF-7 cells were supplemented with 10 μg/mL of human recombinant insulin. Cells were grown in 75 cm² cell culture flasks (Sarstedt, Nümbrecht, Germany) in a humidified atmosphere of CO₂/air (5%/95%) at 37 °C. All the experiments were performed in exponentially growing cultures. Stock solutions of the test compounds were prepared in DMSO and stored in –80 °C for maximum one month. For the cytotoxicity studies, stock solutions of the test compounds were diluted 400-fold with the proper culture medium to obtain the final concentrations. Stock solutions were diluted in a 1:1 ratio with DMSO or the second compound for single compound and combination tests, respectively, so the final concentration of vehicle was constant in each case, and the same stock solution was used in each experiment. For cytotoxicity studies, 2-fold serial dilutions were prepared in the proper medium containing 0.5% DMSO.

To measure the extent of phagocytic activity in LPS-activated microglia, N9 microglial cells were treated with fluorescent polystyrene microbeads (FluoSpheres) measuring 1 μm in diameter (Invitrogen #F13083, ex/em = 580/605 nm). Cells were treated with DHA (10–50 μM) with or without LPS (100 ng/ml) for 6 or 24 h. The LPS was extracted from E. coli of serotype 0111:B4 (Sigma-Aldrich). For control experiments, cells were treated with BSA at concentrations equivalent to that contained in 25 or 50 μM DHA. Seeding flasks used are 75-cm² cell culture flasks (Sarstedt). FluoSpheres were added during the last 3 h of treatment at a concentration of 1 × 10⁶ particles/mL. Cells were washed and fixed in 4 % paraformaldehyde, labeled with Hoechst 33258 (10 μM, 10 min), mounted on glass slides and imaged using a fluorescence microscope. Four fields were imaged per treatment, and a minimum of 20 cells were imaged per field. The number of cells with internalized FluoSpheres was counted.

Four sterilized and moisturized PVA sponges (IVALON^® PVA sponge M-PACT, Eudora, KS, USA) cut to a 6 mm diameter size were implanted subcutaneously after closure of the fascia by creating two small pockets on each side. They were extracted under sterile conditions and transferred to Dulbecco’s modified Eagles media (DMEM, Gibco^TM Fisher Scientific, Karlsruhe, Germany) supplemented with GlutaMax™ (Gibco^TM, Fisher Scientific, Karlsruhe, Germany), 10% v/v fetal calf serum (Pan-Biotech, Aidenbach, Germany) and 0.5% v/v penicillin/streptomycin (Gibco^TM Fisher Scientific, Karlsruhe, Germany). The sponges were maintained overnight in an incubator (37 °C, 5% CO₂). The cells were removed from the sponges by repetitive squeezing and flushing in media the next day. The isolated cells were washed three times and seeded with complete media in 75 cm² cell culture flasks (Sarstedt, Nümbrecht, Germany) for 7 to 10 days. Confluent fibroblasts were passaged by trypsinization (0.25% trypsin-EDTA, Gibco, Karlsruhe, Germany). Fibroblasts (0.5 × 10⁶ total cells) between passage 1 and 4 were used for RNA isolation using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Sponges from different mice of the same group were pooled to obtain a higher cell yield.

Primary mouse astrocytes were prepared from the cortices of newborn C57BL/6 mice (postnatal day (PD) 3–5) as mixed cultures with microglia in Dulbecco’s modified Eagle’s medium (high glutamate) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The growth medium was supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin/streptomycin (Gibco). Fourteen days after seeding in 75 cm² cell culture flasks (Sarstedt AG & Co. KG, Nuembrecht, Germany), the cells were harvested. Culture treatment with PLY and LLO was performed in serum-free medium.
Acute brain slices were prepared from infant (PD 10–14) C57Bl/6 mice via decapitation and vibratome sectioning (Vibroslice NVSL, World Precision Instruments, Berlin, Germany) in artificial CSF continuously oxygenized with carbogen gas (95% O₂, 5% CO₂) at 4 °C. The slices were allowed to adapt in carbogenated Basal Medium of Eagle (Gibco) with 1% penicillin/streptavidin and 1% glucose at 37 °C for 1 h before being challenged with PLY or with LLO in the 5% CO₂-buffered medium (pH = 7.3). In these acute slices, cell lysis never exceeded 7% within 12 h.

Human PSEN1^M146L/WT fibroblasts were obtained from The NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (n = 6) (Camden, NJ, USA), whereas non-mutated control fibroblasts from subjects of the same or another family were obtained from the Coriell Institute (n=2) and from the Uppsala Biobank (n=2) (Uppsala, Sweden) (Table S1) and grown in 75 cm² cell-culture flasks (Sarstedt, Nümbrecht, Germany) in Dulbecco’s modified Eagle’s medium (low glucose, pyruvate, no glutamine, no phenol red) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 U/mL), and 1% GlutaMAX supplement (100×) (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The study was approved by the Regional Ethical Review Board of Uppsala, Sweden, and the Swedish Ethical Review Authority, respectively (protocol numbers 2016/131 and 2020-04131). All experiments were carried out in accordance with the approved protocols.

Human umbilical vein endothelial cells (HUVECs, pooled from several donors) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) were cultured in VascuLife basal medium supplemented with Vascular Endothelial Growth Factor (VEGF) LifeFactors Kit (Lifeline Cell Technology, Frederick, MD, USA), penicillin 0.1 U/ml (Gibco), and streptomycin 100 ng/ml (Gibco). The cells were grown in 75 cm² cell culture flasks (Sarstedt, Nümbrecht, Germany) and kept at 37°C in 95% air and 5% CO₂. Medium was replaced every 48-72 h, and the cells were subcultured when confluent. Cells in passages 5-7 were used.

CCRF-CEM (ECACC 85112105) human Caucasian acute lymphoblastic leukaemia was purchased from ECACC, and A-549 (ATCC CCL 185) human lung carcinoma was purchased from ATCC. The A-549 cell line was cultured in F-12K Medium (Kaighn’s Modification of Ham’s F-12 Medium, Gibco, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco, Waltham, MA, USA), 2 mM L-glutamine, and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). CCRF-CEM was cultured in RPMI 1640 supplemented with 10% foetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, and antibiotics 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in 75 cm² cell culture flasks (Sarstedt, Nümbrecht, Germany) in a humidified atmosphere of CO₂/air (5/95%) at 37 °C. All the experiments were performed in exponentially growing cultures.