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.
Insulin transferrin selenium solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Israel, France
Insulin-transferrin-selenium solution is a specialized cell culture supplement that provides essential components for cell growth and development. It contains insulin, transferrin, and selenium, which are important for maintaining optimal cell health and promoting proliferation in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Tgf β1, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by R&D Systems (3 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Welgene (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
L ascorbic acid, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1a, by Merck Group (2 mentions)
41 protocols using insulin transferrin selenium solution
1
Isolation and Culture of Rat Cardiomyocytes
2
Establishing NAFLD Model in AML12 Cells
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AML12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Welgene, Daegu, Korea), antibiotic/antimycotic solution (Welgene), insulin-transferrin-selenium solution (10 µg/mL insulin, 5.5 µg/mL transferrin, and 5 ng/mL selenium; Invitrogen, Carlsbad, CA, USA), and 40 ng/mL dexamethasone (Sigma‒Aldrich). To establish a NAFLD model using AML12 cells, we used a nonfat BSA-conjugated combination of oleic acid and palmitic acid (OPA) at a ratio of 4:1.
3
Isolation and Expansion of PDAC Cells
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PDAC cells were isolated by mechanical and enzymatic digestion of newborn membranes obtained exclusively from the normal, full-term, postpartum human placenta. PDAC cells were expanded and maintained in the PDAC cell expansion medium until passage 6. PDAC cell expansion medium is composed of Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA) containing 2% (v/v) fetal bovine serum (Invitrogen), 0.001% (w/v) linoleic acid-albumin from bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA), 0.1% (v/v) insulin, transferrin, selenium solution (Invitrogen), 50 μg ml−1 gentamycin (Invitrogen), 100 μM L -ascorbic acid (Sigma-Aldrich), 50 nM dexamethasone (Sigma-Aldrich), 1 ng ml−1 human platelet-human derived growth factor BB (R&D Systems, Minneapolis, MN, USA) and 1 ng ml−1 epidermal growth factor (R&D Systems). PDAC cells were cryopreserved for future use following passage 6 in freezing medium containing 5% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 10% (v/v) human serum albumin (SeraCare Life Sciences, Milford, MA, USA) and 5.5% (w/v) dextran (Hospira, Lake Forest, IL, USA).
4
Immortalized hESC Decidualization Protocol
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The hESC line was immortalized by the retroviral transfection of human telomerase (ATCC CRL-4003) as described by Krikun et al [28 (link)] and was a kind gift from Dr. Haibin Wang (Institute of Zoology, Chinese Academy of Sciences, Beijing, China). The cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 without phenol red (Life Technologies Inc., Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (Biological Industries, Beit Haemek, Israel), 1% (v/v) insulin-transferrin-selenium solution (Invitrogen, Carlsbad, CA, USA), 5 × 10−4 g/L puromycin (Gibco, Grand Island, NY, USA), 5 × 104 U/L penicillin, and 5 × 10−2 g/L streptomycin (Gibco).
To induce in vitro decidualization, the concentration of fetal bovine serum was reduced to 2% (v/v), the cells were treated with 10−3 mM medroxyprogesterone-17-acetate (MPA) (Sigma Chemical Co., St. Louis, MO, USA), and 0.5 mM N6, 2′-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma Chemical Co.) for 7 days, while control samples were treated with 0.1% (v/v) ethanol. As for siRNA transfection, Lipofectamine RNAiMAX Reagent (Invitrogen) was mixed with 5 × 10−5 mM NR5A2 siRNA (Invitrogen), or with a pool of non-targeting control siRNAs. The mixtures were then added to hESCs at 60–80% confluency in 6-well culture plates. After 24 h, the siRNA was removed and the cells were induced to decidualization for 4 days.
To induce in vitro decidualization, the concentration of fetal bovine serum was reduced to 2% (v/v), the cells were treated with 10−3 mM medroxyprogesterone-17-acetate (MPA) (Sigma Chemical Co., St. Louis, MO, USA), and 0.5 mM N6, 2′-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma Chemical Co.) for 7 days, while control samples were treated with 0.1% (v/v) ethanol. As for siRNA transfection, Lipofectamine RNAiMAX Reagent (Invitrogen) was mixed with 5 × 10−5 mM NR5A2 siRNA (Invitrogen), or with a pool of non-targeting control siRNAs. The mixtures were then added to hESCs at 60–80% confluency in 6-well culture plates. After 24 h, the siRNA was removed and the cells were induced to decidualization for 4 days.
5
Sphere Formation Assay for Cancer Stem Cells
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Cells were plated at 1 × 103 cells/well in 6-well ultra-low attachment plates (Corning Inc. Corning, NY, USA). Cell were cultured with serum-free RPMI 1640 (Gibco-Invitrogen, Carlsbad, CA, USA) supplemented with 10 ng/mL fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), 10 ng/mL epidermal growth factor (R&D Systems), and 2.75 ng/mL selenium (insulin-transferrin-selenium solution; Invitrogen). At day 5, spheres larger than 100 µm were counted.
6
Adrenocortical Carcinoma Cell Culture
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H295R cells derived from human adrenocortical carcinoma were purchased from American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 nutrient medium (Wako) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Edina, MN, USA), 1.25 mg/mL BSA (Sigma), 1× insulin-transferrin-selenium solution (Life Technologies), 5.35 mg/mL linoleic acid (Sigma), and 1× penicillin-streptomycin solution (Wako). For angiotensin II-, KCl-, forskolin-, and YM750-induced stimulation, cells were cultured in DMEM (Wako) containing 1% charcoal-treated FBS with penicillin-streptomycin. The culture and stimulation methods were based on previous studies [31 (link),32 (link),33 (link)].
7
Isolation and Culture of Ovarian Cells
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Ovaries from prescribed dpc were collected respectively as described previously. After incubated in 100 μl trypsin solution at 37 °C for 5–10 min, tissues were pipetted up and down for digestion. When the tissues were digested into single-cell suspensions, the digestion reaction was terminated by 20% fetal bovine serum (FBS). The cell suspension was centrifuged at 4 °C, 1000g for 5 min to collect precipitation, then re-suspended in 1 ml PBS and washed thoroughly. The cell suspension was then centrifuged at 4 °C, 1000g, and collected in DMEM/F12 with 4% FBS and 1% modified iTS (Insulin-Transferrin-Selenium Solution; 51500056, Life Technology, USA) supplement. The ovarian cells suspended in DMEM/F12/iTS were cultured in 24-well plates at 37 °C, 5% CO2 for 6–8 h. Plates were gently shaken to flush out the loosely adhered oocytes, and supernatants were collected for centrifugation to collect the oocyte component. The somatic cell component that adhered to the culture plate was recovered by digestion using 0.25% trypsin. Collected cells were cleaned in PBS for further examination.
8
Ovarian Fragment Culture and Analysis
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Thawed ovarian fragments were individually transferred to a Cellstar® 96-well plate U-Bottom (Greiner Bio-One, Italy, 650 185). Each well was filled with 100 μL of Dulbecco’s Modified Eagle Medium (Life Technologies, Belgium, 31053-028) supplemented with 200 mM L-Glutamine (Life Technologies, Belgium, 25030024), 100 μg/ml ascorbic acid, 1 μl/ml Insulin-Transferrin-Selenium solution (Life Technologies, Belgium, 41400-045), 1 μl/ml penicillin-streptomycin (10,000 U/mL; Life Technologies, Belgium, 15140-122), 25 mIU/ml recombinant FSH (Merck, Germany, Gonal-F®) and 5 % normal sheep serum. The tissue fragments were cultured for 2 (D2 condition) or 6 days (D6 condition) at 37 °C in a humidified incubator with 5 % CO2 and 5 % O2 as described by Sanfilippo [28 (link)]. The ovarian punches were fixed in 4 % formaldehyde, embedded in paraffin and cut into 5-μm serial sections for histological analysis.
9
Culturing Human Pluripotent Stem Cells
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The human ESC H9 (WiCell) and IPSC A1ATDR/R (Yusa et al., 2011 (link)) lines were used in this project. Human iPSC line was derived as previously described, under approval by the regional research ethics committee (REC 08/H0311/201). Both hPSCs were cultured on vitronectin XFTM (10 μg/ml, StemCell Technologies)-coated plates and in Essential 8 (E8) chemically defined medium consisting of DMEM/F12 (Gibco), L-ascorbic acid 2-phosphate (1%), insulin-transferrin-selenium solution (2%, Life Technologies), sodium bicarbonate (0.7%), and Penicillin/Streptomycin (1%), freshly supplemented with TGFβ (10 ng/ml, R&D) and FGF2 (12 ng/ml, Qkine) (Chen et al., 2011 (link)). For routine dissociation, cells were incubated with 0.5 μM EDTA (Thermo Fisher Scientific) for 3 min at 37°C seeded in small clumps. Cells were maintained at 37°C in 20% O2, 5% CO2 and medium was replenished every 24 hr.
Authentication of hPSCs was achieved by confirming the expression of pluripotency genes. Cells were routinely confirmed to be mycoplasma free using broth and PCR-based assays. The cell lines are not on the list of commonly misidentified cell lines (International Cell Line Authentication Committee).
Authentication of hPSCs was achieved by confirming the expression of pluripotency genes. Cells were routinely confirmed to be mycoplasma free using broth and PCR-based assays. The cell lines are not on the list of commonly misidentified cell lines (International Cell Line Authentication Committee).
10
Isolation and Culture of Mouse Ovarian Cells
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Mouse ovaries were collected in 1.5 ml tubes and incubated with 0.2 ml of 0.25% trypsin at 37 °C for 2 min and repeated four times. During each incubation, the ovaries were pipetted up and down to digest them into single cell suspensions. A total of 0.2 ml fetal bovine serum was added to each tube to terminate the digestion reaction. The supernatant was removed by centrifugation, and the precipitate was resuspended in 1.2 ml of Dulbecco’s modified Eagle’s medium/Ham F12 nutrient mixture with 10% fetal bovine serum and 1% insulin-transferrin-selenium solution (51500056, Life Technologies). The cell suspension was transferred into 6-well plates and cultured at 37 °C, 5% CO2, and 95% air atmosphere with saturated humidity for 4 to 5 h. The culture medium was collected and centrifuged to collect the oocytes. The adherent cells (somatic cells) were digested with trypsin and collected after centrifugation.
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