Human skin was collected from forensic autopsy specimens that were obtained from the Department of Forensic Medicine of the Kyoto Prefectural University of Medicine. Abdominal skin autopsy specimens from individuals aged 20 to 70 years, which were obtained approximately 1 d after death, were cut into squares with approximate dimensions of 4 cm × 8 cm. Those whose skin was considerably damaged by burning or drowning were excluded. Using the skin-autopsy specimens, an ex vivo model was developed to evaluate the stability of different viruses on the surface of human skin and the effectiveness of different disinfectants against these viruses on the skin [6 ,14 (link)]. The skin from which the panniculus adiposus had been removed was washed with PBS and placed in a culture insert (Corning, Corning, NY, USA) on a membrane with a pore size of 8.0 μm. The culture inserts were placed in six-well plates containing 1.0 mL of DMEM (Sigma-Aldrich).
Culture insert
Manufactured by Merck Group
Sourced in Germany, United States
Culture inserts are a type of laboratory equipment used for cell culture applications. They are designed to provide a controlled and standardized environment for the growth and maintenance of cells in vitro. Culture inserts are typically made of porous membranes that allow for the exchange of nutrients, gases, and waste products between the cells and the surrounding culture medium.
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Lab products found in correlation
Insulin transferrin selenium, by Merck Group (2 mentions)
L ascorbic, by Merck Group (2 mentions)
Albumax 2, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Culture insert, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Culture insert, by Ibidi (1 mentions)
Leibovitz s 15 medium, by Thermo Fisher Scientific (1 mentions)
6 well culture plate, by Corning (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
Hank s buffered salt solution, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal resonant scanner microscope, by Leica camera (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Glucose, by Merck Group (1 mentions)
Heat inactivated horse serum, by Thermo Fisher Scientific (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
17 protocols using culture insert
1
Evaluating Viral Stability on Human Skin
2
In Vitro Cell Migration Assay
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Cell-free gap was created by seeding and culturing cells in Culture-inserts (80241; IBIDI, Martinsried, Germany) until confluent cell monolayer was formed. Cells were treated with 10 μg/mL mitomycin C (M4287; Sigma-Aldrich, Germany) for 2 h before removing Culture-inserts to create cell-free gap. After washing twice with HBSS to remove mitomycin C and detached cells, cells were filled with medium. The cell-free gap was monitored immediately and after 36 h by phase contrast microscope and corresponding pictures were taken.
3
Ovarian Organ Culture Modeling
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Ovaries were dissected from PD4 mice and washed three times in Leibovitz’s-15 medium (Gibco) containing 10% fetal bovine serum plus 1% penicillin–streptomycin before being transferred to culture inserts (Millipore) in a 6-well culture plate (Costar) at 37℃ and 5% CO2. DMEM/F12 (Gibco) supplemented with 5% Insulin-Transferrin-Selenium (Sigma), 1 mg/ml BSA (Sigma), 1 mg/ml Albumax II (Gibco), 100 µM L-ascorbic (Sigma), and 1% penicillin–streptomycin was used as the culture medium. A drop of medium was placed to cover the top of the ovary to prevent drying, and the ovaries were cultured for 4 or 8 days with medium changed every 2 days. Ovaries were treated with control medium (1% DMSO), VCD (30 µM), VCD + MSC-CM (fivefold concentration), or VCD + Fib-CM (fivefold concentration). Appropriate concentrations of recombinant human HGF (100–800 ng/ml), G-CSF (100–800 ng/ml), BDNF (100–800 ng/ml), or HGF neutralizing antibody (0–1 ng/ml) were added directly to the culture medium.
4
Organotypic Hippocampal Slice Culture Protocol
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Organotypic hippocampal slice cultures were prepared using the air/medium interface method63 (link). Briefly, mice were decapitated at P4 and hippocampi were dissected out in cold dissecting medium (Hank’s buffered salt solution 1 × (Gibco), penicillin/streptomycin 1 ×, HEPES (Gibco) 15 mM, glucose (Sigma) 0.5%)). Transverse sections of 300 µm thickness were cut using a tissue chopper. Slices were laid on culture-inserts (Millipore) in pre-warmed six-well plates containing 1.2 mL of maintaining medium (minimal essential medium (MEM) 0.5 × (Gibco), Basal Medium Eagle 25%, horse serum 25%, penicillin/streptomycin 1 × , GlutaMAX 2 mM, glucose 0.65%, sodium bicarbonate 7.5%, ddH2O qsp). Medium was replaced after 24 h and then every 2 days. For culture of Thy1::EGFP; Cx3cr1::CreER; RC::LSL-tdTomato brain slices, 98% Z isomers-OHT was added to the maintaining medium at 0.1 µM during the first 24 h. After preparation, cultures were maintained for up to 21 days in vitro (DIV21) in incubator at 35 °C and 5% CO2. The morphology of microglia was inspected in Cx3cr1::GFP slices at the indicated time-points upon fixation in PFA 4% for 1 h at room temperature, PBS-washed, mounted with mowiol, and imaged on a Leica SP5 confocal resonant scanner microscope with a 63 × /1.4 oil-immersion objective.
5
Organotypic Hippocampal Slice Culture
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Eight-days-old (P8) male and female Thy1-eGFP mouse pups were used for OHSC preparation as described previously (Chai et al., 2014 (link)). In brief, brains were removed from the skull following decapitation under isoflurane anesthesia. The hippocampi were dissected and sliced (400 μm) perpendicular to the longitudinal axis of the hippocampus using a McIlwain tissue chopper. Only slices from the mid region of each hippocampus were used. The slices were placed onto culture inserts (Millipore) and transferred to 6-well plates with 1 ml/well of nutrition medium containing 50% minimal essential medium (MEM), 25% basal medium Eagle (BME), 25% heat-inactivated horse serum (Invitrogen) supplemented with 0.65% glucose and 2 mM glutamate (pH 7.2). OHSC were incubated as static cultures (Stoppini et al., 1991 (link)) in 5% CO2 at 37°C for 7 days in vitro (DIV) before experiments started; the medium was changed every second day.
6
Prdm9 Heterozygous Loss-of-Function on Follicle Survival
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To elucidate the effect of PRDM9 heterozygous loss-of-function variations on follicle survival, we obtained Prdm9 heterozygous knockout mice from Professor Hongbin Liu at Shandong University [15 ]. Ovaries were dissected from the wild-type or heterozygous Prdm9 knockout mice at postnatal 5 days (PD5) and washed three times in Leibovitz’s L-15 medium (Gibco) containing 10% fetal bovine serum and 1% penicillin–streptomycin. Then, the ovaries were transferred into the culture inserts (Millipore) in a 6-well culture plate. The medium containing DMEM/Nutrient Mixture F-12 (DMEM/F12) (Gibco) with 5% insulin–transferrin–selenium (Sigma), 1 mg/mL BSA (Sigma), 1 mg/mL Albumax II (Gibco), 100 µM L-ascorbic (Sigma), and 1% penicillin–streptomycin was used for the in vitro culture. A drop of medium from the well was placed to cover the top of the ovary to prevent drying. Then, the ovaries were cultured at 37°C and 5% CO2 for 8 days, and the medium was changed every 2 days. The 4-vinylcyclohexene diepoxide (VCD) (30 µM), which destroys primordial and primary follicles by accelerating the apoptosis of oocytes [16 (link)], was used to induce exogenous stress to challenge follicle survival.
7
Acute Spinal Cord Slice Incubation
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Acute spinal cord slices from P11-P12 mice were obtained as described in the above paragraph. After incubations in the cutting solutions and regular ACSF, slices were transferred onto culture inserts (Millipore) in 6-well plates and incubated for 2 hr at 37°C in the following solutions: control ACSF (3 mM KCl), high KCl ACSF (12 mM KCl) or 5 mM mannitol ACSF as a control for hypertonic ACSF solution (3-4 lumbar spinal cord slices were incubated per well per condition).
8
Evaluating Cytotoxicity of Drugs in Cell and Tissue Models
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A total of 5 × 104 U118 and U87MG cells per well were seeded into a 6-well plate overnight, and the tissue slices were grown in culture inserts (Millipore) in 6-well plates. Dose–response studies were conducted to determine the suitable concentration of the drugs used in the cell culture and tissue slice experiments. Cells and tissue slices were treated with crizotonib (200 nM [cells]/2 µM [slice cultures]) and temozolomide (5 µM [cells]/50 µM [slice cultures]) for 48 hours. Next, they were incubated with 5 mg/mL of 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide (MTT) at 37°C for four hours. The culture supernatant was then removed and dimethyl sulfoxide (DMSO) (for cells) or 0.1M HCl-isopropyl alcohol (for tissue slices) was added (500 µL/well). Cells were incubated in a shaker at 37°C for 25 minutes until the crystals were completely dissolved. A total of 96-well plates were used to measure the absorbance using the EMax Precision Microplate reader at 570 nm with the reference wavelength set at 620 nm using SoftMax Pro software (Molecular Devices). Optical density was compared by setting the control at 100% and the results were analyzed using Microsoft Office Excel ©. This experiment was performed in triplicate and repeated three times. Calculation of cell viability was described previously.24 (link)
9
Glucose-Stimulated Insulin Secretion Assay
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Glucose stimulated insulin secretion (GSIS) test was performed after 5 days of culture. Native islets and the newly formed spheroids from the different conditions were plated in triplicates in 24-well plates containing culture inserts (Millipore). A preincubation of 1 h in Krebs–Ringer buffered 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4) with 0.1% BSA (KRB solution) containing 2.8 mmol/l glucose was performed. Native islets and spheroids were then exposed for 1 h to a low glucose KRB solution (2.8 mmol/l), followed by 1 h in a high glucose KRB solution (16.7 mmol/l). Supernatants were recovered and insulin concentrations were measured using an ELISA kit (Mercodia, Uppsala, Sweden) for rat insulin. Native islet and spheroid capacity to respond to glucose was expressed as the ratio of insulin concentration in high to low glucose medium, referred to as the stimulation index (SI). Finally, native islets and spheroids were incubated for 1 h in acid ethanol for evaluation of total insulin content. Insulin secretion was further estimated as a percentage of total insulin contents.
10
Multilayer HaCaT Cell Culture
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2×10 5 knockdown HaCaTs and vector control cells were seeded into culture inserts (Millipore), cultured in DMEM supplemented with 10% FCS. After 3 days of submerged culture, cells were raised to the air-liquid interface to produce multilayer structure and fed as required. Samples were collected after ten days of culture. Four independent experiments were undertaken. Sections 5 µm thick were stained with hematoxylin and eosin (H&E) for histological observation. The thickness of the epidermis was measured at three different sites using Image J.
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