Immature oocytes arrested in prophase I (GV oocytes) were obtained from the ovaries of 3–4 week-old ICR female mice. The mice were euthanatized with CO2 and then sacrificed by cervical dislocation, and ovaries were isolated and placed in operation medium (Hepes) with 2.5 μM milrinone and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Oocytes were released from the ovary by puncturing the follicles with a hypodermic needle. Cumulus cells were washed off the cumulus-oocyte complexes (COC) and every 50 isolated denuded oocytes were placed in 100 μl droplets of culture medium under mineral oil (Sigma) in plastic dishes (BD). The culture medium was MEM with 0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM pen/sterep, 3 mg/ml Bovine Serum Albumin (BSA) and 20% FBS (MEM+). Oocytes were cultured at 37.0 °C, 5% O2, 5% CO2 in humidified atmosphere. Prior to IVM (in vitro maturation), all MEM+ include 2.5 μM milrinone to prevent resumption of meiosis.
Plastic dishes
Manufactured by BD
Sourced in United States
Plastic dishes are laboratory equipment designed for various applications in scientific research and testing. They provide a shallow, flat surface made of durable plastic material suitable for a range of laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mineral oil, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Ivis lumina 2 in vivo imaging system, by PerkinElmer (1 mentions)
D luciferin, by PerkinElmer (1 mentions)
Living image software, by PerkinElmer (1 mentions)
Milrinone, by Thermo Fisher Scientific (1 mentions)
7 protocols using plastic dishes
1
Isolation and Culture of Mouse GV Oocytes
2
Mouse Oocyte Isolation and Culture
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Immature oocytes detained in prophase I, that is, germinal vesicle (GV) oocytes, were obtained from the ovaries of 3‐ to 4‐wk‐old ICR female mice. Mice were first anesthetized with CO2, then were euthanized by cervical dislocation, and ovaries were isolated and placed in operation medium (Hepes) with 2.5 nM milrinone and 10% fetal bovine serum (FBS; Thermo Fisher Scientific). Oocytes were dismissed from the ovary by puncturing follicles with a hypodermic needle. Cumulus cells were washed off cumulus–oocyte complexes, and every 50 isolated denuded oocytes were placed in 100 ml droplets of culture medium under mineral oil (MilliporeSigma) in plastic dishes (BD). Culture medium used was minimum essential medium (MEM) supplemented with 0.01 mM EDTA, 0.23 mM sodium pyruvate (Sigma), 0.2 mM penicillin/streptomycin (Gibco), and 3 mg/ml bovine serum albumin (Sigma) that contained 20% FBS (Gibco). Oocytes were grown at 37°C, 5% O2, and 5% CO2 in a humidified atmosphere. Before in vitro maturation, all culture media included 2.5 nM milrinone to prevent the resumption of oocyte meiosis.
3
Bioluminescence Imaging: In Vitro, Ex Vivo, and In Vivo
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In vitro (cells in culture), ex vivo (harvested organs and tissues), and in vivo (living mouse) bioluminescence analysis was performed using the IVIS Lumina II in vivo imaging system (PerkinElmer, Waltham, MA, USA) as previously described.[16 (link)] For in vitro analysis, HCT 116 cells were cultured on plastic dishes (BD, Franklin Lakes, NJ, USA), then incubated with media in the presence of D-luciferin (PerkinElmer) (150 μg/ml) for 5 min, and then analyzed. The procedure was similar for bioptic samples: Tissues were washed in phosphate-buffered saline (PBS), incubated for 5 min in the presence of D-luciferin (150 μg/ml) dissolved in PBS, and then analyzed. For in vivo analysis, animals were anesthetized by intraperitoneal injection of avertin (200 mg/kg). Luciferin dissolved in PBS (150 mg/kg) was also administered intraperitoneally. After 10 min, the animal was put into the detection system, and the signal was acquired in a time range of 1–5 min, depending on signal intensity. Living Image Software (PerkinElmer, Waltham, MA, USA) was used to analyze the signals in manually selected regions of interest. Data were expressed as photons per second per square centimeter per steradian (p/s/cm2/sr).
4
Isolation and Culture of Mouse Oocytes
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Immature oocytes arrested in prophase I (GV oocytes) were obtained from the ovaries of 3–4 week-old female ICR mice. The mice were sacrificed by cervical dislocation and ovaries were isolated and placed in operation medium (Hepes) with 2.5 nM milrinone and 10% fetal bovine serum (FBS) (Gibco). Oocytes were released from the ovary by puncturing the follicles with a hypodermic needle. Cumulus cells were washed off the cumulus-oocyte complexes and 50 isolated denuded oocytes were placed in 100-ul droplets of culture medium under mineral oil in plastic dishes (BD). Oocytes were cultured in MEM + medium (MEM with 0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM penicillin/streptomycin, 3 mg/ml BSA and 20% FBS). Oocytes were cultured at 37.0 °C, in a 5% O2, 5% CO2 humidified atmosphere.
5
Isolation and Culture of Murine GV Oocytes
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Immature oocytes arrested in prophase I (GV oocytes) were obtained from the ovaries of 3-4 week-old ICR female mice in natural estrus. The mice were first euthanatized with CO2 and then sacrificed by cervical dislocation, and ovaries were isolated and placed in operation medium (Hepes) with 2.5 nM milrinone and 10% fetal bovine serum (FBS) (Gibco). Oocytes were released from the ovary by puncturing the follicles with a hypodermic needle. Cumulus cells were washed off the cumulus-oocyte complexes and every 50 Isolated denuded oocytes were placed in 100 ul droplets of culture medium under mineral oil (Sigma) in plastic dishes (BD). The culture medium was MEM+ containing 20% FBS (MEM+ means MEM with 0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM Penicillin/Streptomycin, 3 mg/ml BSA). Oocytes were cultured at 37.0 °C, 5% O2, 5% CO2 in humidified atmosphere. Prior to in vitro maturation (IVM ), all culture medium include 2.5 nM milrinone to prevent resumption of meiosis.
6
Isolation and Culture of Immature Mouse Oocytes
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Fully grown immature oocytes arrested in GV stage were obtained from the ovaries of 3‐4‐week‐old ICR female mice. Oocytes were released from the ovary by puncturing the follicles with a hypodermic needle. Surrounding cumulus cells were removed by repeatedly pipetting. Every 50 isolated denuded oocytes were placed in 100 µL droplets of culture medium under mineral oil in plastic dishes (BD). The culture medium was MEM+ (MEM with 0.01 mmol/L EDTA, 0.23 mmol/L Na‐pyruvate, 0.2 mmol/L penicillin/streptomycin, 3 mg/mL bovine serum albumin [BSA]) containing 20% foetal bovine serum (FBS). Oocytes were cultured at 37.0°C, 5% O2, 5% CO2 in a humidified atmosphere.
7
Isolation and Culture of Rat BMDMs
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Briefly, femurs from rats aged 8 to 10 weeks were flushed and bone marrow derived cells were collected by centrifugation at 450×g for 5 min at 4°C. Cells were then re-suspended in DMEM, supplemented with 10% heat-inactivated FBS and 30% of L-929 M-CSF conditioned medium, and cultured at a density of 1×106 cells/mL in non-tissue culture treated plastic dishes (BD Biosciences, San Jose, CA, USA) in a humidified atmosphere (95% air, 5%CO2) at 37°C. Medium was replaced every 2 days. After 6 days, adherent cells were collected and cell viability was measured by trypan blue. Bone marrow derived macrophages (BMDM) were then diluted in DMEM containing 10% of heat-inactivated FBS and plated in culture dishes for further experiments.
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