Tissue culture plate

Chinese hamster ovary (CHO) cells stably overexpressing APP695 (CHO-2B7 cells) [66 (link)] were grown in Ham’s F-12 medium (Life Technologies) supplemented with 10 % fetal bovine serum and 100 units/ml of penicillin and 100 μg/ml streptomycin. Cells were grown at 37 °C in a humidified atmosphere containing 5 % CO₂ in tissue culture plates (Costar). The cells were harvested at confluence and then utilized for biochemical analyses. Compounds were dissolved in dimethyl sulfoxide (DMSO) and screened in CHO-2B7 cells. The cells were incubated for 16 h in the presence of the compound diluted into OptiMEM-reduced serum medium (Life Technologies, Carlsbad, CA, USA) containing 1 % fetal bovine serum. Compounds used for our study were either purchased from Avanti Polar Lipids, Inc. or synthesized by SAI Life Sciences Ltd. The synthesis schemes of the newly synthesized compounds are demonstrated in Additional file 1.

Kidney epithelial cells from monkey (VERO cells) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Invitrogen, United Kingdom) supplemented with 10% heat inactivated fetal calf serum (Integro B.V., Zaandam, The Netherlands), 1% nonessential amino acids (Gibco), and 1 mM glutamine (Gibco) and incubated in a 37 °C incubator with 5% CO₂. Differentiated VERO cells were prepared by seeding cells from passages 25 to 45 in 12 well tissue culture plates (Costar) at 1.6 × 10⁵ cells/ml in DMEM, with all supplements. The culture medium was replaced every second day. Overnight grown cultures of AUS0004, ΔEFAU004_01209, the complemented strain and the triple mutant were diluted (1:100) and grown at 37 °C to an OD660 of 0.4. Bacteria were harvested by centrifugation and resuspended in DMEM to a concentration of 1 × 107 CFU/ml. For each strain, 1 ml bacterial suspension was added to the wells (100 bacteria per cell). Plates were centrifuged and incubated for 1 h at 37 °C. After incubation, monolayers were rinsed three times with DMEM/EMEM, and cells were lysed with 1% Triton X 100 (Merck, Darmstadt, Germany) in PBS for 5 min at room temperature. The adherent bacteria were quantified by plating serial dilutions on BHI agar plates and counting CFU. The inoculum was plated to determine viable counts. The assay was performed in triplicate and repeated twice

The human colon adenocarcinoma cell-lines Caco-2 (ECACC #86010202), SW620 (ECACC #87051203) and SW480 (ECACC #87092801) were obtained from the European Collection of Authenticated Cell Cultures (Public Health Laboratory Service; Wiltshire, United Kingdom). Intestine-407 cells (ATCC #CCL-6) were obtained from the American Type Culture Collection (LGC Standards: Teddington, United Kingdom). All cell-lines were initially grown in T-75 tissue culture flasks until 80% confluent. Cells were trypsinized, and re-seeded at 1 × 10⁵ cells/well to 24-well tissue culture plates (Corning Costar, High Wycombe, United Kingdom) for infection assays. For luminescence assays, cells were seeded at 9 × 10⁴ per well in 96-well white bottom culture plates (Costar). Parallel cultures in 96-well clear bottom plates were used to assess confluency by light microscopy. All cell-lines were cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% v/v foetal bovine serum (Invitrogen; Paisley, United Kingdom), 100 U/ml penicillin, 100 μg/ml streptomycin and 8 mM glutamine (Sigma Aldrich; Poole, United Kingdom), and maintained at 37°C in a humidified atmosphere of 5% CO₂/95% air for 24–48 h.

SH-SY5Y cells were maintained in T75 culture flasks (Costar, Cambridge, UK) in DMEM/F12 (Labtech, Heathfield, UK) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Labtech, UK), penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 °C in 5% CO₂ and 95% humidity. When cells reached ~80% confluence, they were seeded into 96 well (viability assays) or 24 well (mRNA expression assays) tissue culture plates (Costar, Cambridge, UK) at a density of 5 × 10⁵ cells/cm² and incubated at 37 °C in 5% CO₂ and 95% humidity for 24 h prior to experimentation, or differentiation followed by experimentation. Differentiation was achieved according to the method of de Medeiros et al. [32 (link)]; cells were incubated for 1 day in DMEM/F12 with 10% serum followed by 3 days in DMEM/F12 with 1% serum plus 10 μM Retinoic acid (RA) and then 3 days in DMEM/F12 with 1% serum plus 10 μM RA plus 50 ng/mL bone derived neurotrophic factor (BDNF) (Abcam, Cambridge, UK).

Cell culture reagents were purchased from Gibco-Invitrogen, while tissue culture plates and other plastic materials were obtained from Corning. Salts and buffers were obtained from Sigma-Aldrich, as well as thioflavin T (ThT), black Sudan B, Congo-red, SDS, [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] tetrazolium (MTT), tunicamycin (Tum), and lipopolysaccharides O111:B4 (LPS). l-α-phosphatidylcholine (PC), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA) were obtained from Avanti Polar Lipids.

Cell culture reagents were purchased from Thermo Fisher (Carlsbad, CA, USA), while tissue culture plates and other plastic materials were obtained from Corning Inc. (Corning, NY, USA). Salts and buffers were obtained from Sigma-Aldrich (St. Luis, MI, USA), as well as Thioflavin T (ThT), black Sudan B, Congo red, sodium dodecyl sulfate (SDS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). l-α-phosphatidylcholine (PC), l-α-phosphatidylserine (PS), l-α-phosphatidyl-ethanolamine (PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (LPA), 1-palmitoyl-2-oleoyl-sn-glycerol (POPG), and cholesterol were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Antibodies anti-XBP1s and anti-BiP/GRP78 were purchased from Abcam (Cambridge, UK) and anti-β-actin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-PDI was donated by Dr. Marco A. Ramos Ibarra.