Organotypic hippocampal slice cultures were prepared from P5 C57BL/6 wild-type mice of both sex according to.37 (link) In brief, mice were decapitated and the hippocampus was extracted and sliced into coronal sections of 300 μm thickness. The slices were placed on small pieces of Polytetrafluoroethylene (PTFE) membrane, pore size 0.45 μm (Millipore, # FHLC01300) and cultured on cell culture insert of 0.4 μm pore size (Millipore, # PICM03050) placed in a 6-well plate and then incubated at 37°C with 5% CO2 for 18 to 23 days in vitro (DIV). The inhibitor mix containing Ara-C (Sigma-Aldrich, #C6645), Uridine (Sigma-Aldrich, #U3750) and 5-Fluoro-2′-desoxyuridine (Sigma-Aldrich #F0503), was added to a final concentration of 3 μM on the third day and the medium was exchanged three times per week.
Fhlc01300
Manufactured by Merck Group
The FHLC01300 is a laboratory equipment product manufactured by Merck Group. It serves as a core function for laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6jrj mice, by Janvier Labs (2 mentions)
F0503, by Merck Group (1 mentions)
Picm03050, by Merck Group (1 mentions)
C6645, by Merck Group (1 mentions)
U3750, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
5 protocols using fhlc01300
1
Organotypic Hippocampal Slice Culture
2
Organotypic Hippocampal Slices for Alzheimer's
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Organotypic hippocampal slices were prepared from 5- to 7-day-old C57bl6JRj mice (Janvier Labs). Brains were quickly dissected to retrieve hippocampi, and then, both hemispheres were sliced in 400-μm sections and kept into the slicing medium (97.5% Earles’ Balanced Salt solution (EBSS) and 2.5% EBSS-Hepes). Slices were transferred on sterile hydrophilic membrane Millicell discs (FHLC01300, Millipore) placed in semi-porous cell culture inserts (0.4 μm, Millipore) containing the culture medium (Minimum Essential Medium Eagle + Glutamax-1 (50%), EBSS (18%), EBSS/D-glucose 13% (5%), penicillin-streptomycin (5000 U/ml, 1%), horse serum (25%), and nystatin (10,000 U/ml, 0.06%)). Two hours after plating, slices were infected with lentiviruses encoding for green, APPwt, and APPswe proteins, respectively. Slices were kept at 37 °C, with CO2 (5%) for 9 days before imaging analysis. For sitagliptin treatment, slices were incubated for 24 h at a concentration of 100 μM.
3
Organotypic Hippocampal Slice Preparation
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Organotypic hippocampal slices preparations were achieved on C57bl6JRj mice (Janvier Labs, Le Genest Saint-Isle, France) 5 to 7 days after birth. Brains were quickly dissected out to retrieve hippocampi from both hemispheres, that were then sliced onto 400 µm sections and kept into slicing medium [Earles’ Balanced Salt solution (EBSS; 97.5%) and EBSS-HEPES (2.5%)]. Slices were transferred on sterile hydrophilic membrane millicell discs (Millipore, FHLC01300) placed in semiporous cell culture inserts (Millipore, 0.4 µm) containing culture medium (Minimum Essential Medium Eagle (MEM) + Glutamax-1 (50%), EBSS (18%), EBSS (13%)/D-glucose (5%), penicillin–streptomycin 5000U/ml (1%), Horse serum (25%) and Nystatin 10000U/ml (0.06%). Slices were infected 2 h after plating with 2 µl of lentiviruses encoding for lenti-Green (virus titer: 2.44 × 1010), lenti-Green-APPwt, (virus titer: 2.21 × 1010) or lenti-Green-APPswe, (virus titer: 2.21 × 1010). Slices were kept at 37 °C, 5% CO2 for 9 days before treatments and imaging experiments.
4
Acute Hippocampal Slice Culture
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Hippocampal slices were obtained from 5–7-day-old mice of either sex and cultured on cell culture inserts with porous membranes. Mouse pups were decapitated and the hippocampus was isolated while the brain was submerged in ice-cold sterile filtered HBSS without Ca2+ and Mg2+ (Gibco, 14175-053) supplemented with 10 mM glucose, using a stereo microscope. Hippocampi were cut into 350-µm thick slices and placed on round porous membranes with 4-mm diameter (PTFE membrane; Merck, FHLC01300) that were placed on cell culture inserts with a porous membrane (Millicell, PICM0RG50) for interface culture. The inserts with the slices were placed in dishes (Greiner, 627161) with 1 ml culture medium. We adapted the medium recipe during the course of experiments, as quality of cultures deteriorated with the same nominal composition. We found that 78.5% minimum essential medium (Gibco, 11095-080), 15% heat-inactivated horse serum (Gibco, 26050070), 2% B27 supplement (Gibco, 0080085SA), 2.5% 1 M HEPES (Sigma, M3375-100G), 1.5% 0.2 M GlutaMax supplement (Gibco, 35050-061) and 0.5% 0.05 M ascorbic acid (Sigma, A5960-25G), with additional 1 mM CaCl2 and 1 mM MgSO4 produced satisfactory results and incubated at 37 °C and 5% CO2. The medium was changed the day after preparation and then every 3–4 d.
5
Culturing Hippocampal Slices from Mice
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Hippocampal slices were obtained from 5-7 days old mice of either sex and cultured on cell culture inserts with porous MEMbranes. Mouse pups were decapitated and the hippocampus was isolated while the brain was submerged in ice cold sterile filtered HBSS without Ca 2+ and Mg 2+ (Gibco, #14175-053) supplemented with 10 mM glucose, using a stereo microscope. Hippocampi were cut into 350 µm thick slices and placed on round porous MEMbranes with 4 mm diameter (PTFE MEMbrane, Merck, #FHLC01300), which have been placed on cell culture inserts with a porous MEMbrane (Millicell, #PICM0RG50) for interface culture. The inserts with the slices were placed in dishes (Greiner, #627161) with 1 ml of culture media. We adapted the media recipe during the course of experiments, as quality of cultures deteriorated with the same nominal composition. We found 78.5% Minimum Essential Medium (MEM, Gibco, #11095-080), 15% heat-inactivated horse serum (Gibco, #26050070), 2% B27 supplement (Gibco, #0080085SA), 2.5% 1 M HEPES (Sigma, #M3375-100G), 1.5% 0.2 M GlutaMax supplement (Gibco, #35050-061), 0.5% 0.05 M ascorbic acid (Sigma, #A5960-25G), with additional 1 mM CaCl2 and 1 mM MgSO4 to produce satisfactory results, and incubated at 37 °C and 5% CO2. The medium was changed the day after preparation and then every 3-4 days.
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