Fifteen hours after the hCG injection, mice were sacrificed by CO2 inhalation and cervical dislocation. Ovulated oocytes were immediately recovered from the ampulla region of the fallopian tube into EmbryoMax® M2 buffered collection medium (M2: EMD Millipore, Temecula, CA) with 200 μM 1-methyl-3-(2-methylpropyl)-7H-purine-2,6-dione (IBMX: Sigma-Aldrich) to prevent any recovered GV stage oocytes from resuming meiosis before evaluation. Cumulus cells were removed by quickly pipetting the oocytes through a small bore (100 μm) glass pipette in M2+IBMX and 300 μg/ml hyaluronidase (Sigma-Aldrich) and then immediately washed into fresh M2+IBMX. Oocytes were evaluated for meiotic resumption using the same criteria as the monkey and those with MI and MII configuration were reported as a combined GVBD rate, comparable to the reporting format in Li, et al. [9 (link)].
Embryomax
Manufactured by Merck Group
Sourced in United States, Germany
EmbryoMax is a versatile lab equipment designed for embryo culture and handling. It provides a controlled environment to support the growth and development of embryos.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (3 mentions)
M2 medium, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
2 beta mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Esgro, by Merck Group (2 mentions)
Tissue culture treated polystyrene dishes, by Corning (2 mentions)
Ksom aa, by Merck Group (2 mentions)
Intergonan, by MSD (2 mentions)
Intergonan, by MSD animal health (2 mentions)
Chorulon, by MSD animal health (2 mentions)
8w10e arrays, by Ibidi (2 mentions)
Alpha minimal essential medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Nucleoside, by Merck Group (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Optimem medium with glutamax, by Thermo Fisher Scientific (1 mentions)
Sigenome smartpool, by Horizon Discovery (1 mentions)
31 protocols using embryomax
1
Recovering Ovulated Oocytes from Mice
2
Vitrified Oocyte Warming and Embryo Culture
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The oocytes were warmed within 5 days following vitrification. The warming solutions consisted of PBS supplemented with 20% FBS and three different concentrations of sucrose (1, 0.5, and 0.25 M). The Cryotop device was directly immersed in a 37 °C warming solution including 1 M sucrose for 1 minute, followed by immersion in a warming solution containing 0.5 M sucrose for 3 minutes. Finally, the device was transferred to a warming solution with 0.25 M sucrose for 3 minutes. The recovered oocytes were then moved to a solution of PBS with 20% FBS and subjected to 3 minutes of incubation for washing. After the washing step, the survival rate of the vitrified-warmed oocytes was evaluated.
Surviving oocytes were incubated in 1 mL of HTF with 10% SSS, including 10 μL of epididymal sperm suspension (0.3 to 2 million/mL), at 37.0 °C in a 5% carbon dioxide (CO2) humidified air environment. Inseminated oocytes were cultured for 24 hours, and two-cell embryos were obtained on the following day (considered day 1).
The two-cell embryos were washed twice using pipetting and then cultured in potassium simplex optimized medium (KSOM; EmbryoMax; EMD Millipore) supplemented with 10% SSS. The culture conditions were maintained at 37 °C with 5% CO2 in a humidified air environment.
Surviving oocytes were incubated in 1 mL of HTF with 10% SSS, including 10 μL of epididymal sperm suspension (0.3 to 2 million/mL), at 37.0 °C in a 5% carbon dioxide (CO2) humidified air environment. Inseminated oocytes were cultured for 24 hours, and two-cell embryos were obtained on the following day (considered day 1).
The two-cell embryos were washed twice using pipetting and then cultured in potassium simplex optimized medium (KSOM; EmbryoMax; EMD Millipore) supplemented with 10% SSS. The culture conditions were maintained at 37 °C with 5% CO2 in a humidified air environment.
3
Culturing Mouse Uterine Epithelial Cells
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Mouse uterine epithelial cells (MUECs) were purchased from Creative Bioarray (CSC-C9063J, New York, NY, USA) and cultured in SuperCult® Complete Epithelial Cell Medium (ECBM, Creative Bioarray) containing 2% fetal bovine serum (FBS, Creative Bioarray, USA), 1% L-glutamine (Creative Bioarray) and 1% antibiotic-antimitotic solution (Creative Bioarray). Cells were maintained in a humidified 5% CO2 environment at 37 °C. Trypsinized cells were seeded at a density of 25 × 103 cells/cm2 on a 12 well plate coated with 0.1% gelatin solution (EmbryoMax®, Merck Millipore). Culture medium was replenished every 2 days until cells reached 70% confluence.
To prepare conditioned medium, ECBM medium was replaced with fresh KSOM when MUECs were 70% confluent. To ensure complete removal of traces of ECBM and FBS, cells were washed twice in Dulbecco Phosphate’s Buffered Saline (DPBS, Sigma Aldrich). Cells were then incubated for 24 h in a humidified 5% CO2 environment at 37 °C. Conditioned medium was sterilized by passage in a 0.2 µm-pore-diameter Millipore filter and immediately used.
To prepare conditioned medium, ECBM medium was replaced with fresh KSOM when MUECs were 70% confluent. To ensure complete removal of traces of ECBM and FBS, cells were washed twice in Dulbecco Phosphate’s Buffered Saline (DPBS, Sigma Aldrich). Cells were then incubated for 24 h in a humidified 5% CO2 environment at 37 °C. Conditioned medium was sterilized by passage in a 0.2 µm-pore-diameter Millipore filter and immediately used.
4
Arsenite-Induced Cytotoxicity and Hepatogenesis
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A human UC-MSC cell line was obtained from ATCC and cultured in alpha minimal essential medium (Gibco, USA) supplement with 10 % fetal bovine serum (FBS, EmbryoMax®, Merck Millipore, USA), 1% of 200 mM L -Glutamine (Gibco), and 1 % of 10,000 U Penicillin/Streptomycin (Gibco). For cytotoxicity, DNA damage, and DNA damage-related gene experiments, UC-MSCs were treated with varying concentrations of sodium arsenite (NaAsO2) for 24 h (0–5 µM). For hepatocyte differentiation studies, UC-MSCs were treated at 0–0.5 µM NaAsO2 for 21 days during differentiation into hepatocytes, comparison to the control.
5
Senescence Induction in Cell Lines
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1 mg/ml SAICAr (Carbo Synth) was added to the cells for 96 hrs, to mimic ADSL depletion. 60 µM nucleosides (100X Embryomax, Merck Millipore) were added from the first silencing to the end at 1X in the culture medium. MRT00252040 (kindly provided by Simon Osborne, LifeArc, London, UK) dissolved in DMSO was used at 2 µM and MTX (Millipore Sigma) at 4 µM as described in (14) . ATM inhibitor (KU-55933; Selleckem) was used at 5 mM for 24 hrs before fixation. Doxorubicin (Millipore Sigma) was used as positive control for senescence at 1 ug/ml for 6 days.
6
Embryo Thawing and Handling Protocol
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The embryos were thawed according to the procedure described by the European Mouse Mutant Archive (EMMA) (EMMA-The European mouse mutant archive, 2013b; Hagn et al., 2007) . Briefly, the straw was thawed at room temperature (RT), and then the embryos were rinsed in 4 successive M2 medium drops and placed in KSOM medium (Embry-oMax®, Merck Millipore, Darmstadt, Germany) .
The number of thawed embryos used for each experiment performed in this study is presented in Table 1.
The number of thawed embryos used for each experiment performed in this study is presented in Table 1.
7
Differentiation of HE cells to NK cells
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Following a 3-day subculture with glutamine or in glutamine-free medium with DMK (1.75 mM, SIGMA) and/or Nucleosides (Cytidine: 7.3 mg/L; Guanosine: 8.5 mg/L; Uridine: 7.3 mg/L; Adenosine: 8 mg/L; Thymidine: 2.4 mg/L; EmbryoMax, Millipore), HE cells were collected with StemPro Accutase Cell Dissociation Reagent and seeded onto OP9-DL1 stroma (80% confluent). The OP9-DL1 murine cell line was kindly provided by Dr. Ewa Sitnicka-Quinn (Lund University, Sweden) and its authentication was described in Renoux et al.41 (link). The protocol used to induce NK cell differentiation was described previously by Renoux et al.41 (link). Briefly, co-cultures were kept in OP9 medium, consisting of OptiMEM medium with Glutamax (Invitrogen) with 10% FCS, 1% penicillin–streptomycin solution (Thermo Fisher Scientific) and 1% 2-mercaptoethanol (Invitrogen) with SCF (10 ng/ml), FLT3-L (10 ng/ml), IL-2 (5 ng/ml), IL-7 (5 ng/ml, first 15 days only) and IL-15 (10 ng/ml). Cells were passaged onto new OP9-DL1 stroma every week. At day 35, cells were stained for human CD45 and CD56 markers and analyzed on a BD LSRFortessa.
8
Knockdown and Chemical Inhibition Assays
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For knockdown experiments, cells were transfected 24–96 h before sample collection with the indicated siRNA using RNAiMax transfection reagent (Life Technologies) according to the manufacturer's instructions: siLUC (100–200 nM; 5′-GGUACGCGGAAUACUUCGAdTdT-3′), siFZR1, siRad51, siAPE1, siTDG (100–200 nM siGENOME SMARTpool Dharmacon). The following reagents were used to treat ES cells for the indicated time at the indicated final concentrations before collection: ATM inhibitor (KU55933, Kudos; 8 h, 10 μM); ATR inhibitor (ETP-46464, provided by O. Fernandez-Capetillo, CNIO, Madrid; 8 h, 5 μM; VE-821, Selleckchem; 8 h, 10 μM); PARP inhibitor (Olaparib, Selleckchem; 24 h, 10 μM); Reducing/scavenging agent: (N-acetylcysteine, Sigma; 10 h, 10 mM); Transcription inhibitors (Cordycepin, Sigma; 100 min, 50 μM; Alpha-amanitin, kindly provided by P. Janscak, 3–6 h, 20 μM); Ape1 inhibitor (Methoxyamine hydrochloride, Sigma; 10 h, 1 μM); CDK4/6 inhibitor (LY2835219, Selleckchem; 4 h, 1 μM); Cdc7 inhibitors (PHA-767491, Sigma; 8 h, 10 μM; XL-413, kindly provided by C. Santocanale, 4 h, 10 μM); CDK1 inhibitor (RO-3306, Sigma; 10 h, 10 μM); PLK inhibitor (BI-6727, Selleckchem; 4 h, 500 nM); Nucleosides (EmbryoMax, Millipore; 24 h, 5 ×). Roscovitine (Seliciclib, Selleckchem; 8 h, 20 μM).
9
Superovulation and Zygote Collection in Mice
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Oocyte and embryo collection were performed according to standard procedures [37 ]. Four- to six-week-old females were superovulated by an intraperitoneal (i.p.) injection of pregnant mare serum gonadotropin (PMSG, 5 IU) followed by human chorionic gonadotropin (hCG; 7.5 IU) 48 h later. Fertilized eggs were harvested at 20 h post-hCG in M2 media (Sigma-Aldrich, Ref. M7167, St. Louis, MO, USA. Cumulus cells were removed by thorough pipetting in M2 media containing hyaluronidase. Good-quality zygotes were thoroughly washed three times in clean dishes containing M2 media and KSOM (Embryomax®; Millipore, Ref. MR-020P, Burlington, MA, USA, and cultured in vitro in KSOM at 37 °C, 5% CO2, 90% relative humidity.
All procedures were carried out under Project License PI29/08, approved by the in-house Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 on the protection of animals used for experimental and other scientific purposes.
All procedures were carried out under Project License PI29/08, approved by the in-house Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609 on the protection of animals used for experimental and other scientific purposes.
10
Culturing Adherent Fibroblasts in Gelatin
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Fibroblasts were resuspended in media (DMEM supplemented with 10% FBS) and seeded into a plate (Falcon) coated with EmbryoMax™ ultrapure water with 0.1% gelatin (Millipore). All cells were maintained under sterile conditions in a humidified incubator under 5% CO2 at 37 °C. A phase-contrast microscope (Leica) was used to image cells.
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