Cumulus-oocyte complexes (COCs) with ≥3 layers of granulosa cells were isolated from 4 to 6 week-old female mice by puncturing the ovary with a syringe needle. After granulosa cells were removed, GV oocytes were washed with the M2 medium (M7167, Sigma-Aldrich, United States) and transferred into the M16 medium supplemented with 10% FBS (10100147, Gibco, United States), 50 mIU/ml FSH (5925-FS-010, R&D Systems, United States) and 1 μg/ml 17β-estradiol (E8875, Sigma-Aldrich, United States). After being cultured for 12 h at 37°C in the atmosphere with 5% CO2, the oocytes with the first polar body were transferred to HTF medium (MR-070, Millipore, United States) and incubated with capacitated spermatozoa at 37°C in the atmosphere with 5% CO2. After 6 h, fertilized oocytes (with two pronuclei) were examined. All oocytes were then transferred into a pre-balanced KSOM medium (MR-121, Millipore, United States) at 37°C in a humidified atmosphere with 5% CO2. After 18h, two-cell embryos were examined. After another 72 h, blastocysts were examined.
Mr 070
Manufactured by Merck Group
Sourced in United States
The MR-070 is a laboratory instrument designed for specialized magnetic resonance measurements. It is a versatile tool that can be used for various analytical and research applications. The core function of the MR-070 is to generate and detect magnetic resonance signals, which can provide valuable information about the properties and behavior of materials and samples under investigation.
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Lab products found in correlation
6 protocols using mr 070
1
Mouse Oocyte Maturation and Fertilization
2
Evaluating Mouse Oocyte Developmental Potential
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To evaluate the capability of oocyte developmental, we carried out IVF assays according to our previous protocols (Han et al., 2018 (link)). Sperm, collected from aged 10–20 weeks male mice, were left to capacitate for 1 h in HTF fertilization medium (MR070; Millipore) supplemented with 10 mg/ml bovine serum albumin, and then co‐incubated with MII oocytes matured in vitro in HTF drops at 37°C for 5 h. Following fertilization, presumptive zygotes were washed in order to remove excess sperm. Finally, zygotes were transferred into KSOM medium (MR106D; Millipore) and cultured up to the blastocyst stage at 37°C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2.
3
Mouse Sperm Fertilization and ZP Binding
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In vitro fertilization using mouse spermatozoa was performed as described previously (27 (link)). To prepare cumulus- and ZP-free oocytes, cumulus-oocyte complexes were treated with 0.3 mg/mL hyaluronidase (H4272, Sigma-Aldrich, MO, USA). Then, for ZP removal, the cumulus-free oocytes were treated with 1 mg/mL collagenase (C1639, Sigma-Aldrich, MO, USA) as described previously (28 (link)). Two hours of preincubated spermatozoa were inseminated with the differently treated oocytes using Toyoda-Yokoyama-Hoshi (TYH) or Human Tubal Fluid (HTF) mediums for in vitro fertilization. To analyze sperm ZP-binding ability, cumulus-free oocytes were incubated with capacitated spermatozoa in HTF medium (MR-070, Sigma-Aldrich) for 30 min before observation. The bound spermatozoa were fixed with 0.25% glutaraldehyde (Nacalai Tesque, Kyoto, Japan) and were counted. To prepare destabilized ZP oocytes and assess sperm ZP penetration ability, CARD medium and FERTIUP were used under manufacturing protocols (Kyudo Company, Saga, Japan).
4
Mouse Sperm Fertilization Protocol
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The 8-week male mouse was sacrificed, and the epididymis was obtained. The epididymis was cut 3–4 times and transferred to HTF medium (Sigma, MR-070) to harvest matured sperms. After 1 h of sperm capacitation, sperms and oocytes were co-incubated for 4h ∼ 6h at 37 °C, 5% CO2 in the air. Then sperms were washed, and the fertilized oocyte was transferred to the KOSM medium (Sigma, MR-101-D). The zygote was going to start cleavage within 12h.
5
Epididymal Sperm Motility Analysis
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Human tubal fluid (HTF) solution (MR-070, Sigma-Aldrich) was pre-equilibrated for 2 h in a constant temperature incubator at 37 °C. The cauda epididymis was separated and placed into 500 μl of HTF. After 15 min of swim-out in an incubator at 37 °C, sperm motility in the supernatant was analyzed using a sperm analysis system (CASA system version12, CEROS, Hamilton Thorne Research). More than 400 sperm were counted for each sample.
6
Sperm Characteristics in Diabetic Mice
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Mature sperm in the mouse cauda epididymis were prepared as described before.21 (link) Two caudal epididymis samples were placed into human tubal fluid (HTF) medium (cat# MR-070, Sigma–Aldrich). Then, we slightly cut the cauda of the epididymis into three pieces for incubation at 37°C for 5 min. We performed sperm concentration and motility analyses using the computer-assisted semen analysis system (CASA; WLJY-9000, Beijing Weili Co., Ltd., Beijing, China) according to the laboratory manual of the World Health Organization.3 Parameters including sperm concentration (×106 ml-1), rapid progressive motility (grade A; in %), progressive motility (grade A + B; in %), curve-line velocity (VCL; in μm s−1), straight-line velocity (VSL; in μm s−1), linearity (LIN; in %), average path velocity (VAP; in μm s−1), amplitude of lateral head displacement (ALH; in μm), and straightness (STR; in %) were evaluated. At least 200 sperm were counted for each assay. Seven, 8, 7, and 6 mice in the db/m, db/db, Dapa, and Ex groups were used for semen analysis, respectively.
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