Millicell pcf

OHSCs were conducted in a slightly modified manner from the previous report [16 (link)]. Young adult male B6 mice (5-week-old) were anesthetized by Avertin (0.4 g/kg body weight, Sigma, St. Louis, MO, USA) and decapitated. The hippocampi were rapidly dissected in ice-cold artificial cerebrospinal fluid (aCSF) consisting of the following (in mM): NaCl 118, KCl 2.5, MgSO₄ 3, NaH₂PO₄ 1.1, NaHCO₃ 26, CaCl₂ 1, and glucose 11 (all reagents from Sigma), bubbled with 95% O₂/5% CO₂. Subsequently, coronal slices (350 μm thick) were cut with a vibratome (VT1200S, Leica, Wetzlar, Germany). The sliced hippocampi were transferred onto sterile 0.4 μm porous membrane confetti (Millicell-PCF, Millipore, Ireland) and cultured with medium consisting of 50% MEM, 25% horse serum, 18% HBSS, 4 mM L-glutamine, 12 mM glucose, 4.5 mM NaHCO₃, 20 mM sucrose, 100 U/mL penicillin, and 100 mg/mL streptomycin (all reagents from Gibco or Sigma) in a humidified 5% CO₂ atmosphere at 37°C. The medium was changed three times a week. On day in vitro (DIV) 5, slices were treated with MP (10 μM) or vehicle (DMSO) for 12 hr. The cell death level of the slices was then examined by PI staining. Furthermore, the BDNF level was examined to evaluate the neuroprotective effect of MP treatment on the hippocampal slices.

Human embryonic kidney cell line HEK293 was cultured in high glucose DMEM containing 10% FBS and 1% penicillin‐streptomycin. MpkCCD_c14 cells were cultured in defined media containing 2% FBS, antibiotics and other hormones, as previously described (Bens et al. 1999; Itani et al. 2005). A549 and H441 human lung epithelial cells and HT‐29 colonic epithelial cells were cultured as previously described (Mick et al. 2001; Itani et al. 2002).
MpkCCD_c14 cells were transduced with each of three different GIPZ shRNAmir lentiviral particles specific to Sgk1 (clone ID: V3LHS_328980, V3LHS_636735, V3LHS_636738) or with non‐silencing shRNAmir control lentivirus according to manufacturer's instructions. Cells were maintained under puromycin selection for isolation of single cell clones. Individual clones were expanded to generate stable cell lines expressing shRNAs. Wild type mpkCCD_c14 cells or stable mpkCCD_c14 GIPZ lentiviral clones were seeded on collagen‐coated transwell filters (12‐mm‐diameter Millicell‐PCF, Millipore) and short‐circuit measurements (I_sc) were measured in culture media at 37°C in Ussing chambers as described previously (Raikwar et al. 2012).

HEK293T (ATCC, CRL-11268), HEK293 (ATCC, CRL-1573), A549 (ATCC, CCL-185), and HeLa cells (ATCC, CCL-2) were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10% fetal bovine serum (FBS; GIBCO) at 37°C with 5% CO₂. Human bronchial epithelial Calu-3 cells were cultured in DMEM-nutrient mixture F-12 Hams (1:1) supplemented with 1% MEM nonessential amino acids solution (GIBCO), 1% GlutaMAX (GIBCO), and 10% FBS (GIBCO) at 37°C with 5% CO₂. Calu-3 cells were seeded at a density of 5 × 10⁵ cells/cm² on semipermeable membrane inserts (0.6 cm²; Millicell-PCF; Millipore). After 3 to 4 weeks of culture for differentiation, cells with transepithelial electrical resistance (TEER) values over 600 V/cm² were used for HBoV infection.