A total of 15 male SD rats aged 5–8 weeks and weighing 150–200 g were housed at 25 °C with 30% humidity and a 12 h light/dark cycle, with free access to water and food. At the beginning of the study, the animals were sacrificed by cervical dislocation. The ventricular tissues were cut into sections approximately 1 mm3 using ophthalmic scissors, then were washed twice in phosphate buffered saline without calcium or magnesium ions. The tissue pieces were moved into 10 mL centrifuge tubes for further digestion by trypsin and type II collagenase. The CMs were collected by centrifugation (TDZ4-WS, Xiangyi, Shanghai, China) at 500 × g for 5 min at room temperature. The isolated cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cell culture plates were precoated with 10 μg/mL of fibronectin and the cells were cultured in medium (CM media) containing Dulbecco’s-modified eagle medium (DMEM/F-12) with 2-hydroxyethyl (HEPES) (Invitrogen, Shanghai, China), 10% foetal bovine serum, 1% antibiotic-antimycotic solution (Invitrogen), 3 mM pyruvic acid, 2 mg/mL bovine serum albumin, 100 μg/mL ampicillin and insulin-transferrin-selenium solution (20 ng/mL, 11 ng/mL, and 13.4 pg/mL, respectively; Invitrogen), 5 μg/mL linoleic acid and 100 μM ascorbic acid (Nguyen et al. 2012 (link)). For all experiments, cells were cultured at 5 × 104 cells/mL unless otherwise stated.
Pyruvic acid
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Pyruvic acid is a organic chemical compound that serves as a key intermediate in various metabolic pathways. It is a colorless, odorless liquid that is highly soluble in water and widely utilized in scientific research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Thermo Fisher Scientific (3 mentions)
Glucose, by Merck Group (2 mentions)
Progesterone, by Merck Group (2 mentions)
Human bfgf, by Thermo Fisher Scientific (2 mentions)
Murine leukemia inhibitory factor, by ProSpec (2 mentions)
Mem vitamins, by Welgene (2 mentions)
Dl lactic acid, by Merck Group (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
β estradiol, by Merck Group (2 mentions)
Human egf, by Thermo Fisher Scientific (2 mentions)
Human gdnf, by Thermo Fisher Scientific (2 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Welgene (2 mentions)
19 protocols using pyruvic acid
1
Isolation and Culture of Rat Cardiomyocytes
2
Characterization of HER2-expressing Breast Cancer Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines expressing pcDNA3, pcDNA3-HER2 were cultured and characterized using methods previously described [51 (link)]. Briefly, MCF7 cells engineered to express HER2 or contain the empty vector pcDNA were maintained in MEM supplemented with essential and non-essential amino acids (Invitrogen), Pyruvic acid, 10% glutamine and 10% FBS. Cells were harvested after they reached 80% confluence. Breast cancer cell lines BT474 and MDA453, which were originally derived from HER2-positive breast cancers that exhibited amplified HER2 [54 (link), 55 (link)] were purchased from ATCC and cultured using the recommended protocols.
3
Culturing Cell Lines with Mitochondrial Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells and B82 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. HeLa cells and B82 cells devoid of endogenous mitochondrial genomes (HeLa ρ0 cells and B82 ρ0 cells) were kindly provided by Dr Kazuto Nakata (Tsukuba University). HeLa ρ0 cells and B82 ρ0 cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS, pyruvic acid (Invitrogen, final concentration 10 μM) and uridine (Sigma, final concentration 100 μg/m). Hybridoma cells that produce ms2i6A antibody were cultured in GIT medium (Wako) at 37°C and 5% CO2.
4
Cell Culture and Differentiation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PC12 cells were maintained and differentiated with NGF as previously described [18 (link)]. For NGF treatment, cells were grown in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 1% heat-inactivated horse serum (Sigma-Aldrich), penicillin/streptomycin (Thermo Fisher Scientific) and 50 ng/ml recombinant human β-NGF (Alomone Labs, Jerusalem, Israel) for 7-8 days in a 7,5% CO2 atmosphere at 37°C. Medium was changed every other day and before transfection.
HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (all from Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37°C.
Rat primary cortical cultures were prepared as previously reported [19 (link)]. Briefly, cortex from embryonic (E18) Sprague-Dawley rats were dissected out, dissociated and plated at a density of 250 cells/mm2 on poly-L-lysine-coated (Sigma-Aldrich) coverslips or at a density of 700 cells/mm2 on poly-L-lysine-coated plates. Neurons were maintained in neurobasal medium with B27 and 2mM GlutaMAX (all from Gibco). However, neurons plated on coverslips were initially seeded with MEM medium supplemented with 100 mM pyruvic acid (Gibco), 20% glucose (Sigma-Aldrich) and 10% heat-inactivated horse serum.
HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (all from Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37°C.
Rat primary cortical cultures were prepared as previously reported [19 (link)]. Briefly, cortex from embryonic (E18) Sprague-Dawley rats were dissected out, dissociated and plated at a density of 250 cells/mm2 on poly-L-lysine-coated (Sigma-Aldrich) coverslips or at a density of 700 cells/mm2 on poly-L-lysine-coated plates. Neurons were maintained in neurobasal medium with B27 and 2mM GlutaMAX (all from Gibco). However, neurons plated on coverslips were initially seeded with MEM medium supplemented with 100 mM pyruvic acid (Gibco), 20% glucose (Sigma-Aldrich) and 10% heat-inactivated horse serum.
5
Establishment of Transgenic SSCs from Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Establishment of SSCs from Oct4-GFP/LacZ and Oct4-GFP transgenic mice (C57BL/6 background) was described previously (Ko et al. 2009 (link); Kanatsu-Shinohara et al. 2011 (link); Han et al. 2012 (link)). SSC medium for expansion was composed of StemPro-34 SFM (Gibco, Carlsbad, CA, USA) with the following supplements: StemPro (Gibco), 1× N2 (Gibco), 6 mg/ml d-(+)-glucose (Gibco), 30 mg/ml pyruvic acid (Gibco), 1 μl/ml DL-lactic acid (Sigma-Aldrich, Saint Louis, MO, USA), 5 mg/ml bovine serum albumin (BSA; Gibco), 1% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), 50 μM β-mercaptoethanol (Gibco), 1×penicillin/streptomycin (Welgene, Gyeongsan, Korea), 1× minimal essential medium (MEM) with non-essential amino acids (Gibco), 1× MEM vitamins (Welgene), 30 ng/ml β-estradiol (Sigma), 60 ng/ml progesterone (Sigma), 20 ng/ml human EGF (Peprotech, Rocky Hill, NJ, USA), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec, Rehovot, Israel).
6
Establishment of SSCs from Oct4-GFP/LacZ Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Establishment of SSCs from Oct4-GFP/LacZ transgenic mice (C57BL/6 background) was described previously (Ko et al., 2009 (link); 2010 (link); 2012 (link)). SSC medium for expansion was composed of StemPro-34 SFM (Gibco) with the following supplements: StemPro supplement (Gibco), 1× N2 supplement (Gibco), 6 mg/ml d-(+)-glucose (Gibco), 30 mg/ml pyruvic acid (Gibco), 1 μl/ml DL-lactic acid (Sigma-Aldrich), 5 mg/ml bovine serum albumin (BSA; Gibco), 1% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 50 μM β-mercaptoethanol (Gibco), 1×penicillin/streptomycin (Welgene), 1× minimal essential medium (MEM) non-essential amino acids (Gibco), 1× MEM vitamins (Welgene), 30 ng/ml β-estradiol (Sigma-Aldrich), 60 ng/ml progesterone (Sigma-Aldrich), 20 ng/ml human EGF (Peprotech), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec).
7
Neuronal Cell Culture and Treatments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cortices and hippocampi from newborn (P0-P1) wild-type and Col6a1−/− mice were dissociated in trypsin (0.8 mg/ml, Sigma) for 10 min at 37°C. Digestion was blocked by a solution containing trypsin inhibitor (6.3 μg/ml, Sigma) and DNase I (40 μg/ml, Invitrogen). Dissociated cells were plated at a density of 2.5×105 cells per cm2 well on glass coverslips or on plastic coated with either poly-L-lysine (100 μg/ml), collagen I (Sigma), or purified native murine collagen VI [12 (link)]. The culture medium consisted of MEM (GIBCO) containing glucose (20 mM), L-glutamine (0.5 mM), N2 supplement (1%), B27 supplement (0.5%), biotin (0.875 mg/l), pyruvic acid (1 mM), penicillin (25 μg/ml) streptomycin (25 μg/ml), fungizone (50 μg/ml) and horse serum (10%, Gibco). Cytosine-β-d-arabinofuranoside (3 μM) was added 24 hr after plating. Cultures were grown for 7 days, and the last day cells were subjected to different treatment conditions for 4.5 hr. The following treatments were used: DMEM (Gibco) without serum; DMEM without serum, in the presence of 3-methyladenine (10 mM, Sigma); DMEM without serum, in the presence of chloroquine (50 μM, Sigma); DMEM without serum, in the presence of rapamycin (100 nM, LC-Laboratories). Cells were fixed in 4% paraformaldehyde.
8
Fetal Gonad Organoid Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fetal gonads were obtained from E12.5 embryos, and divided into 2 pieces and cultured on 24-well plates (corning) with 1 mL of growth medium in each well at 37 • C with 5% CO 2 for 24-96 h. The growth medium was composed of α-minimal essential medium (α-MEM) (Gibco, 12571063), supplemented with 10% heatinactivated fetal bovine serum (BI, 04-001-1ACS), 0.23 mM pyruvic acid (Gibco, 11360070), 10 mIU/mL FSH (Merckserono), 100 mIU/mL penicillin G, and 100 mg/mL streptomycin sulfate (Gibco, 10378016). The gonad tissues were treated with Retinoic acid (R&D, 0695/50) at a concentration of 1 µM, Recombinant human/mouse Wnt5a Protein (R&D, 645-WN-010) at a concentration of 350 ng/mL and combination respectively.
The somatic cells were obtained from E12.5 wildtype female mesonephros, which were cultured in each well of a 6-well plate with 2 mL of Dulbecco's modified Eagle's medium (Gibco, 11995065) containing 10% (v/v) fetal bovine serum (FBS) (BI, 04-001-1ACS) and 1% Penicillin/Streptomycin (Gibco, 10378016) in a humidified incubator at 37 • C with 5% CO 2 for 24-96 h. The somatic cells were treated with LY294002 (Sigma, 10 µM) for 24 h to inhibit PI3K activity, or treated with Wnt3a (R&D, 5036WN, 100 ng/mL) for 24 h to induce Wnt/β-catenin signaling.
The somatic cells were obtained from E12.5 wildtype female mesonephros, which were cultured in each well of a 6-well plate with 2 mL of Dulbecco's modified Eagle's medium (Gibco, 11995065) containing 10% (v/v) fetal bovine serum (FBS) (BI, 04-001-1ACS) and 1% Penicillin/Streptomycin (Gibco, 10378016) in a humidified incubator at 37 • C with 5% CO 2 for 24-96 h. The somatic cells were treated with LY294002 (Sigma, 10 µM) for 24 h to inhibit PI3K activity, or treated with Wnt3a (R&D, 5036WN, 100 ng/mL) for 24 h to induce Wnt/β-catenin signaling.
9
Primary Hippocampal Neuron Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hippocampal cultures were prepared from P0 male and female Sprague Dawley rats as previously described (Tonini et al., 2013) (link) and in accordance with the UK Animals (Scientific Procedures) Act 1986; protocols were reviewed and approved by the UCL Animal Welfare and Ethical Review Body. Briefly, after dissection of the hippocampus, 2.5% trypsin (Invitrogen, Paisley, UK) was applied and the tissue mechanically dissociated using polished glass Pasteur pipettes. Cells were plated onto poly-D-lysine (100 µg/ml, Sigma-Aldrich, Dorset, UK)-coated glass coverslips at a density of 18,500 -23,500 viable cells/cm 2 and allowed to settle in Minimal Essential Medium supplemented with 10 % Horse Serum, 1 mM pyruvic acid, 2 mM glutamine (all from Gibco, Paisley, UK), and 0.6% glucose (Sigma-Aldrich, Dorset, UK) in a humidified incubator at 37˚C in 5% CO 2 . This attachment solution was replaced after 4-14h with Neurobasal medium (Gibco, Paisley, UK) containing 2 % B27 supplement (Gibco, Paisley, UK), 2 mM glutamine (Gibco, Paisley, UK), 0.6 % glucose and penicillin/streptomycin (100 U/ml/100 µg/ml, Sigma-Aldrich, Dorset, UK). Cultures were grown for 10-12 days before conducting imaging or electrophysiological experiments and the growth medium was replaced every 5-7 days.
10
Anaerobic Bacterial Growth Optimization
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!