Earle s balanced salt solution ebss

Organotypic cerebellum brain slice cultures were prepared according to previously published protocols [35 (link),36 (link),37 (link),38 (link)]. In brief, cerebella were derived from P10–P12 mice and separated from the rest of the brain. The cerebellum was dissected in ice-cold minimum essential media (MEM) supplemented with Glutamax (Gibco) until taken for cutting sagittal slices of 300 µm thickness using a McIlwain Tissue chopper. The separated slices were transferred onto humidified sterile culture membrane inserts with pore sizes of 0.4 µm (Merck Millipore, Watford, UK). Tissue slices on membrane inserts were cultured on a liquid layer of medium containing 50% MEM media (Gibco), 25% HS, 25% Earle’s Balanced Salt Solution (EBSS) (Gibco) supplemented with 130 mM D-glucose (Sigma) and 1% P/S. The cultures were first maintained at 37 °C in 5% CO₂ for 24 h prior to being used for in vitro experiments. After 24 h, the medium bathing the tissue was replaced with serum-free medium, into which treatments were added including vitamin K1 (100 µM) or warfarin (25 µM). After 72 h incubation, the culture medium was analysed for released Gas6 protein by ELISA assays for both total mouse Gas6 and Gla-Gas6 (below).

The retinal tissues were separated from the enucleated eyeballs of newborn Sprague-Dawley rats on postnatal days 1 to 4 and incubated in precooled calcium-free and magnesium-free Earle’s Balanced Salt Solution (EBSS; Gibco, Grand Island, NY) and Hank’s Balanced Salt Solution (Life Technologies, Grand Island, NY) containing 5 mg/mL papain, 0.24 mg/mL l-cysteine, and 10 U/mL DNase І for 30 min. Then, an ovomucoid solution containing 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO), 0.1% ovomucoid (Sigma-Aldrich), and 1% DNase І (4 mg/mL, Sigma-Aldrich) in minimum essential medium (MEM; Gibco) was subsequently used to fully quench any residual papain activity. After centrifugation at 200×g for 10 min, the cells were resuspended in MEM containing 0.5 mg/mL BSA and then the cell suspension was filtered through the mesh filter (pore size 40 μm; BD Falcon, Franklin Lakes, NJ) to yield a single cell suspension. The procedures were conducted at room temperature in a laminar flow hood.

Acetone, ethanol, and IPA were purchased from Millipore. The SU-8 and SU-8 developers were purchased from MicroChem. The amorphous fluoroplastic solution was purchased from Chemours Company. Pluronic F127 was purchased from Sigma-Aldrich (Oakville, ON, USA). Silicone oil (1 Cst) was purchased from Clearco, USA. Fetal bovine serum (FBS), PBS, collagenase II, Hank’s Balanced Salt Solution (HBSS), Earle’s balanced salt solution (EBSS), DMEM/F12, Glutamax, HEPES, and 1:50 B27 were purchased from Gibco. Cis-diammineplatinum (II) dichloride, EP, Y27632, dexamethasone, penicillin/streptomycin, N-acetyl-l-cysteine, nicotinamide, insulin, hydrocortisone, cholera toxin, and hyaluronidase were purchased from Sigma. Wzb117 was purchased from Selleckchem. Recombinant human EGF, recombinant human FGF10, and recombinant human HGF were purchased from Peprotech. RBC lysis buffer, EthD-1, erythrocyte lysate, and Cell Tracker™ Green CMFDA Dye were purchased from Invitrogen. StemMACS iPS-Brew XF medium was purchased from Miltenyl Biotec (USA). Dimethyl sulfoxide, Sor, Reg, Apa, Len, and DNase I were purchased from Solarbio. Forskolin and A8301 were purchased from Tocris.

Primary antibodies and dilutions used for Western blotting were as follows: ATG3 C-ter (1:1,000, Abcam, ab108282), ATG3 N-ter (1:1,000, Abcam, ab108251), ATG4B (1:1,000, Cell Signaling Technology, no. 5299), β-actin (1:4,000, Sigma, A1978), FLAG biotin-conjugated (1:1,000, Sigma, F9291), GFP (1:2,000, Clontech, no. 632381), LC3A/B (1:500, Cell Signaling Technology, no. 12741), LC3B (1:1,000, Sigma, L7543), SQSTM1 (1:1,000, Sigma, P0057), vinculin (1:1,000, Abcam, ab129002).
Secondary antibodies for Western blotting with chemiluminescence-based detection were as follows: goat anti-rabbit/anti-mouse IgG, HRP-linked (1:5,000, Cell Signaling Technology, no. 7074/no. 7076). For fluorescence-based detection, the following were used: IRDye 800CW goat anti-rabbit/anti-mouse (1:15,000 with 0.02% SDS, LI-COR no. 926-32211/926-32212); IRDye 680LT goat anti-rabbit/anti-mouse (1:25,000 with 0.02% SDS, LI-COR no. 926-68021/926-68020).
Torin1 was purchased from Merck-Millipore (no. 475991), and bafilomycin A₁ from Streptomyces griseus was purchased from Sigma (B1793); both were dissolved in DMSO. Recombinant GST–ATG4B WT and C74S were produced and purified as described previously (65 (link)). Earle's balanced salt solution (EBSS) containing calcium and magnesium was purchased from Thermo Fisher Scientific (no. 24010-043).

Hippocampal neurons were obtained from dissection of P0 pups of either ApoE3 or ApoE4 mice as previously described58 (link). ApoE3 or ApoE4 neurons at DIV10 were incubated with either 500 nM of Aβ42 or scrambled Aβ42 24 h prior to starvation. The Aβ42 concentration used in this study was comparable to other studies using primary neurons44 (link)53 (link). On the day of the experiment, the Neurobasal media (Life Technologies) was removed from the wells and the neurons were starved in Earle’s Balanced Salt Solution (EBSS) (Life Technologies) for 2 hours. After starvation, 2 nM of insulin (Sigma Aldrich) was added into the respective wells and incubated for 30 minutes. Following the acute insulin treatment, the neurons were collected for Western Blot analysis.