Neuroblastoma SH-SY5Y cells with and without stable transfection of human APP770 were maintained in Eagle’s minimum essential medium/Ham’s F-12 medium (Thermo Scientific, Waltham, MA) supplemented with 1% nonessential amino acids and 10% fetal bovine serum. The murine microglial BV-2 cell line was purchased from Istituto Nazionale per la Ricerca sul Cancro (Genova, Italy) and cultured in RPMI 1640 (Thermo Scientific) supplemented with 10% foetal bovine serum and l -glutamine. Primary neuronal cultures were prepared from cerebral cortices of embryonic day 15 mice using a dissociation solution (Sumitomo Bakelite, Tokyo, Japan). The cells (5 × 105/cm2) were plated on polyethyleneimine-coated dishes and cultured for 7 days in neurobasal medium with 25 mM KCl, 2 mM glutamine and B27 supplement (Thermo Scientific). For Transwell cultures, APP-expressing SH-SY5Y cells (5 × 105/cm2) cultured on 24-well plate inserts (0.5 µm pore; Corning, NY) and BV-2 cells (1 × 106) placed below the inserts were treated for 24 h with 10 µM GlcCer or ceramides in Eagle’s minimum essential medium/Ham’s F-12 medium.
24 well plate inserts
Manufactured by Corning
Sourced in United States
The 24-well plate inserts are a laboratory equipment designed to fit into standard 24-well culture plates. They provide a contained environment for growing and culturing cells or performing other experimental procedures within a multi-well plate setup.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dissociation solution, by Sumitomo Bakelite (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 protocols using 24 well plate inserts
1
Neuroblastoma Cell Co-culture System
2
Transwell Macrophage-EpSC Coculture Protocol
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A transwell system was used to evaluate the influence of polarized macrophages on EpSCs according to our previous method [9 (link)]. Macrophages were cultured on 24-well plate inserts (1-mm pore size, Corning, NY, USA) with EpSCs preplated on the bottom wells. In brief, the polarized macrophages (M0, M1 or M2) were washed with PBS, resuspended in B27 medium, and subsequently added to the inserts at an initial seeding density of 5×105 cells per insert. The inserts were placed on a 24-well plate that contained EpSCs at 5×105 cells per well (bottom well). The bottom wells were precoated with poly-L-lysine (PLL; Sigma-Aldrich,). The cells were cultured in medium (B27 [w/o vitamin A, 1×], L-glutamine [2 mM], penicillin [100 U/ml], streptomycin [100 µg/ml] [all from Invitrogen, CA, USA], and partricin [0.5 µg/ml; Sigma-Aldrich]) for 3~7 days. The macrophages in the inserts were changed every 3 days. The EpSCs were collected and used for immunostaining and western blotting assays according to the experimental scheme.
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