Following intraperitoneal euthanasia with pentobarbitol, fresh brain tissue was rapidly extracted from P10 male rats, placed in ice cold dissection media, and sectioned into 300 μm slices using a Mcllwain tissue chopper (Ted Pella, Redding, CA, USA). These slices were plated onto 30 mm cell culture inserts (Millipore Sigma, Burlington, MA, USA) in nontreated 6-well plates (USA Scientific, Orlando, FL, USA). Slices were then incubated at 37 °C in 1 mL of 5% slice culture medium (SCM) at 37 °C and 5% CO2 to recover from acute slicing. SCM medium with 5% horse serum consists of 180 mL HBSS (Gibco), 20 mL horse serum, 200 mL MEM (Gibco, Dublin, Ireland), 4 mL PenStrep, and 4 mL GlutaMax (Gibco). Days in vitro (DIV) 0 was defined as the day of brain extraction and slicing. At DIV1, the media was exchanged with fresh 5% SCM.
30 mm cell culture insert
Manufactured by Merck Group
Sourced in United States, Germany
The 30 mm cell culture inserts are a lab equipment product designed for cell culture applications. They provide a platform for growing and studying cells in vitro. The inserts are made of a semi-permeable membrane that allows for the exchange of nutrients, gases, and waste between the cells and the surrounding media.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Mcllwain tissue chopper, by Ted Pella (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Lumicycle apparatus, by Actimetrics (1 mentions)
Microtome blade, by Leica Biosystems (1 mentions)
Techno glass, by AGC Techno Glass (1 mentions)
Alpha mem glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 protocols using 30 mm cell culture insert
1
Acute Brain Slice Preparation and Culture
2
Circadian Rhythm Monitoring in Tendon
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LumiCycle apparatus (Actimetrics, Wilmette, IL, USA) was used for real-time quantitative bioluminescence recording. Tail tendons, 10 mg, were placed in 30-mm cell culture inserts (0.4 µm pore size; MilliporeSigma, Burlington, MA, USA) inside 35-mm dishes in recording medium [DMEM without phenol-red (D2902; MilliporeSigma), supplemented with 4 g/L glucose, 5% fetal calf serum, HEPES, sodium bicarbonate, and 0.1 mM luciferin]. iTTFs were seeded 24 h, as previously described, prior to drug treatments. Dexamethasone (100 nM) was added for 30 min to synchronize the circadian rhythms before plating in recording medium. Baseline subtraction was carried out using a 24-h moving mean. Amplitude was calculated as peak-trough difference in bioluminescence of the first and second peak, as indicated, using baseline-subtracted data.
3
Ex Vivo Ovarian Tissue Slicing and Culture
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Ovaries from 4-week-old mice were sliced at a thickness of several hundred micrometers using a microtome blade (Leica Biosystems, Nussloch, Germany) under a stereoscopic microscope (Leica Biosystems). Ovarian tissue slices were
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
4
P0 Mouse Ovary Tissue Culture
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On postnatal day 0 (P0), Oog1pro3.9/R26-H2B-mCherry female mouse ovaries were used for ovarian tissue culture experiments. Whole P0 mouse ovaries were cultured in a 30 mm
cell culture insert (Merck Millipore, Darmstadt, Germany). The culture conditions were maintained and detailed methods were performed as reported previously [19 ].
cell culture insert (Merck Millipore, Darmstadt, Germany). The culture conditions were maintained and detailed methods were performed as reported previously [19 ].
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