HEK293T, Vero E6, and IPEC-J2 cell lines were preserved in our laboratory. HEK293T and Vero E6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Yeasen, Shanghai, China) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphozone) (Gibco, Waltham, MA, USA). IPEC-J2 cells were grown in a DMEM/nutrient mixture F-12 (DMEM/F12) (Gibco, Waltham, MA, USA) supplemented with 10% FBS and antibiotics. All cells were cultured at 37 °C and 5% CO2 atmosphere. The PEDV strain ZJ15XS0101 (GenBank accession No. KX550281.1) isolated from a clinically diseased pig was propagated and titrated in Vero E6 cells [32 (link)]. Mouse anti-PEDV-N monoclonal antibody (mAb), mouse anti-PEDV-S1 mAb, and rabbit anti-DNAJA3 polyclonal antibody (pAb) were prepared in our laboratory. Mouse anti-β-actin mAb was purchased from Beyotime (Shanghai, China), and the mAbs to GST, HA, or Flag tag from ABclonal (Wuhan, China).
F 12 dmem f12
Manufactured by Thermo Fisher Scientific
Sourced in United States
F-12 (DMEM/F12) is a cell culture medium commonly used in the laboratory. It is a basal medium that provides essential nutrients and supports the growth and maintenance of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Forskolin, by MedChemExpress (1 mentions)
Chir99021, by MedChemExpress (1 mentions)
Repsox, by MedChemExpress (1 mentions)
Ldn 193189, by MedChemExpress (1 mentions)
Gentamicin solution, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Bt 20, by American Type Culture Collection (1 mentions)
26 gauge needle, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Arpe 19, by American Type Culture Collection (1 mentions)
Arpe 19, by Thermo Fisher Scientific (1 mentions)
P creb, by Bioworld Technology (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
11 protocols using f 12 dmem f12
1
PEDV Propagation and Antibody Production in Cell Lines
2
Screening Stem Cell Signaling Modulators
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A screening of small molecules from the MedChemExpress stem cell signaling library (catalogue No. HY-LO17) was performed using a basic cocktail containing forskolin (5 μM), (MedChemExpress, Monmouth Junction, NJ, USA), CHIR99021 (2 μM), (MedChemExpress, Monmouth Junction, NJ, USA), RepSox (2 μM) (MedChemExpress Monmouth Junction, NJ, USA), and LDN193189 (0.5 μM), (MedChemExpress, Monmouth Junction, NJ, USA) in Dulbecco’s modified Eagle medium: F12 (DMEM: F12) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing N2 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the only supplement. Each of the small molecules from the library was added to the basic cocktail at a final concentration of 5 μM and investigated for its ability to generate a neuronal-like morphology and Tuj1+ cells.
3
In vitro and in vivo assays protocol
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High purity chemicals and reagents were purchased from commercial sources, and all dilutions were prepared immediately before use. For in vitro assays, Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12), Roswell Parki Memorial Institute (RPMI-1640), Leibovitz’s (L-15), fetal bovine serum (FBS), Epidermal Growth Factor (EGF), insulin and antibiotic gentamicin solution were purchased from (Gibco®). The 3-[4,5-dimethylthiazole-2—yl]2,5-diphenyltetrazolium bromide (MTT), CAS 57360-69-7, dimethylsufoxide (DMSO), CAS 67-68-5 and hydrocortisone were purchased from Sigma-Aldrich®. Tamoxifen Citrate (Sandoz) was gently donated by the Cancer Hospital from Federal University of Uberlandia.
For in vivo assays, Doxorubicin hydrochloride (DOX), commercial name Fauldoxo® (CAS 25316-40-9, batch 19B1091, Laboratório Industrial Brasileiro de Biologia e Síntese - Libbs, São Paulo – Brazil) was used as positive control at 0.4 mM. This concentration was based on previous studies that demonstrated the induction of epithelial tumors in D. melanogaster by DOX [44 (link), 50 –53 (link)]. Tween 80 (CAS 9005-65-6) at 1% (v/v) was used as negative control and for dilution of the compounds.
For in vivo assays, Doxorubicin hydrochloride (DOX), commercial name Fauldoxo® (CAS 25316-40-9, batch 19B1091, Laboratório Industrial Brasileiro de Biologia e Síntese - Libbs, São Paulo – Brazil) was used as positive control at 0.4 mM. This concentration was based on previous studies that demonstrated the induction of epithelial tumors in D. melanogaster by DOX [44 (link), 50 –53 (link)]. Tween 80 (CAS 9005-65-6) at 1% (v/v) was used as negative control and for dilution of the compounds.
4
Culturing Breast Cancer Cell Lines
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The human breast cancer cell lines BT-474, SKBR3, BT-20, MDA-MB-231, MCF-7, and T-47D were obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% streptomycin-penicillin in a humidified atmosphere at 37 °C with 5% CO2.The immortalized mammary epithelial cell line MCF10A were obtained from ATCC and cells were cultured in Dulbecco’s Modified Eagle Medium: F-12(DMEM/F-12) (Gibco, USA) supplemented with 5% horse serum, 50 ng/ml epidermal growth factor (EGF), 10 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.1 mg/ml cholera toxin and 1% streptomycin-penicillin at 37 °C in an atmosphere of 5% CO2 and 95% air.
5
Purification of Tachyzoites from T. gondii RH Strain
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Tachyzoites of T. gondii RH strain were maintained as described previously, with some modifications [24 (link)]. Briefly, human retinal pigment epithelial cells, ARPE-19 (ATCC, Manassas, Virginia, USA), were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) with F12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic–antimycotics (all from Gibco, Grand Island, New York, USA).
ARPE-19 cells were infected with RH strain of T. gondii using a multiplicity of infection (MOI) of 5 and incubated at 37°C and 5% CO2 for 2–3 days. Following spontaneous host cell rupture, parasites and host cell debris were washed in cold PBS. The final pellet was resuspended in cold DMEM and then passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, Massachusetts, USA).
ARPE-19 cells were infected with RH strain of T. gondii using a multiplicity of infection (MOI) of 5 and incubated at 37°C and 5% CO2 for 2–3 days. Following spontaneous host cell rupture, parasites and host cell debris were washed in cold PBS. The final pellet was resuspended in cold DMEM and then passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, Massachusetts, USA).
6
Sheep Ovarian Granulosa Cell Isolation
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During the peak breeding season (August to October), healthy sheep ovaries from animals aged 1 to 1.5 years were sourced from a local abattoir in Shihezi, Xinjiang Uygur Autonomous Region, China. Mature dominant follicles were carefully selected, their follicular fluid aspirated and collected into Petri dishes containing (Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 [DMEM/F12] (Gibco, France) medium. Oocytes were meticulously picked using a mouth pipette. The GCs were then transferred to erythrocyte lysis buffer to eliminate any red blood cells. The pelleted cells were washed twice with DMEM/F12 medium, cultured into Petri dishes enriched with 10% fetal bovine serum [FBS] (Gibco, France), 100 IU/mL penicillin, and 100 μg/mL streptomycin) aseptic culture at 37°C in a 5% CO2 atmosphere. After 48 h, non-adherent cells were removed by gentle medium replacement.
7
Radix Polygalae Compound Identification
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3,6’-Disinapoyl sucrose and TSFA were isolated from Radix Polygalae, identified by a combination of spectral methods (UV, IR, MS, and NMR) and purified by HPLC (purity >90%). U0126 and LY294002 were purchased from Selleck Chemicals LLC (Houston, TX, USA). Dulbecco’s modified Eagle’s medium (nutrient mixture F-12; DMEM/F-12) was obtained from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT, USA). Polyclonal rabbit antibodies against BDNF, CREB, pCREB, CRTC1, and β-actin were all provided by Bioworld Technology (Atlanta, GA, USA) or Abcam (Burlingame, CA, USA).
8
Isolation and Culture of Adipose-Derived Mesenchymal Stem Cells
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Human adipose tissue was obtained from patients who had undergone lipoplasty, and all enrolled patients signed the informed consent form to indicate their agreement and consent to the use of their adipose tissue in this study. The liposuction sites were bilateral thighs and buttocks. The adipose tissue was rinsed three times with phosphate-buffered saline (PBS, Solarbio, Beijing, China), and then 0.1% type I collagenase (Sigma–Aldrich, St. Louis, MO, USA) was used to digest the adipose tissue for 50 min. Then, the stromal vascular fraction was filtered through a 70 μm porous filter (Millipore, Hayward, CA, USA) after centrifugation at 2000 rpm for 10 min. After that, AD-MSCs were resuspended in Dulbecco’s modified Eagle medium: F-12 (DMEM/F-12, Gibco BRL, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco BRL, NY, USA) and 1% penicillin–streptomycin (Solarbio, Beijing, China) at 37 °C in an incubator with 5% carbon dioxide (CO2). The medium was changed every two days. Cells in passage four were used in this experiment.
9
Melatonin Modulates ER Stress Pathways
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Dulbecco's modified Eagle medium, a nutrient mixture of F-12 (DMEM/F12), fetal bovine serum (FBS), penicillin and streptomycin, was purchased from GIBCO-BRL (Gaithersburg, MD, USA). Presto Blue reagent, TRIZOL reagent, and the Superscript First-Strand Synthesis System were purchased from Invitrogen (Carlsbad, CA, USA). The following antibodies were used for western blotting: anti-Bip, anti-CHOP, anti-PERK, anti-phospho-PERK, and anti-phospho-eIF2α (Cell Signaling Technology, Danvers, MA, USA) anti-ATF6 and anti-XBP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiphospho-IRE1α and anti-caspase-12 (Abcam, Cambridge, UK) and anti-β-actin (1: 2000 Millipore, Bedford, MA, USA). Melatonin and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
10
Isolation and Culture of Murine and Human Cell Lines
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Mesenchymal stem cells (MSCs) were isolated from bone marrow of C57BL/6 female mice according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. C57BL/6 MSCs were also purchased from Thermo Fisher Scientific Life Sciences (Waltham, MA, http://www.thermofisher.com ). MSCs stably expressing the LacZ gene (β‐Gal‐MSCs) via a lentiviral system, as previously described [11 ]. Cell lines were maintained in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F‐12 (DMEM/F12) (Thermo Fisher), supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher), 2 mM l ‐glutamine, and 1% penicillin/streptomycin. Human 14‐week male embryonal lung cell line MRC‐5 (BCRC‐60023) was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan, http://www.bcrc.firdi.org.tw ). MRC‐5 cells were maintained in Eagle's Minimal Essential Medium (MEM) (Thermo Fisher), supplemented with 10% FBS and 1% penicillin/streptomycin and were incubated at 37°C in a 5% CO2 incubator. Mouse macrophage‐like cell line RAW264.7 (BCRC‐60001) was purchased from Bioresource Collection and Research Center. Cell lines were maintained in DMEM containing 10% heat‐inactivated FBS.
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