Human embryonic kidney 293T cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (100 units/mL penicillin–100 µg/mL streptomycin [Pen-Strep]; Gibco). MDCK cells were maintained in Minimum Essential Medium (Gibco) supplemented with 10% FBS, Pen-Strep, L-glutamine (Gibco), sodium bicarbonate (Corning) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco). Cell lines were maintained at 37 °C with 5% CO2.
4 2 hydroxyethyl 1 piperazineethanesulfonic acid
Manufactured by Thermo Fisher Scientific
Sourced in United States
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is a chemical compound commonly used as a buffer solution in biochemical and cell culture applications. It maintains a stable pH environment for various biological processes and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Penicillin, by Thermo Fisher Scientific (10 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Pen strep, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
K562 cell, by American Type Culture Collection (2 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
44 protocols using 4 2 hydroxyethyl 1 piperazineethanesulfonic acid
1
Cell Line Maintenance Protocols
2
Immunomodulatory Effects of Calcineurin Inhibition
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RPMI-1640, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid were purchased from Gibco (Grand Island, NY, USA). Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China). FK506, MFN2, and β-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methyl-thiazolyl-tetrazolium (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IL-4 and interferon (IFN)-γ were obtained from Biosource (Worcester, MA, USA). Fluo-3/AM and pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Total RNA isolation and reverse transcription systems were purchased from Promega (Madison, WI, USA). The Biomol Green Calcineurin Assay kit was purchased from Biomol (Plymouth Meeting, PA, USA). Nuclear extract and TransAM NFAT kits were obtained from Active Motif (Carlsbad, CA, USA). Nondenaturing lysis buffer and protease inhibitor cocktail were purchased from Applygen Technologies Inc., (Beijing, China). An Amersham enhanced chemiluminescence (ECL) Advance Western Blotting Detection kit was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).
3
Thawing and Enumeration of Frozen PBMCs
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Frozen PBMC vials were removed from liquid nitrogen storage tanks and placed into a 37 °C water bath. Once samples were thawed and removed from the water bath, the vials were wiped down with 70% isopropyl alcohol and the cell suspensions transferred using plastic transfer pipettes (Fisher Brand) to a 15 mL round bottom tube (Corning). Roswell Park Memorial Institute Medium (RPMI [Gibco]) with 10% Fetal Calf Serum (FCS [Omega]), 1% L-glutamine (Gibco), 100 U/mL Penicillin-Streptomycin (Gibco), 12.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES [Gibco]) buffer was added dropwise to each round bottom tube to gradually dilute the cell suspension. Between dropwise additions of culture medium, the cell suspensions were agitated to ensure homogeneity. Cells were centrifuged at 300g for 10 min at room temperature, then resuspended in 1 mL of 10% FCS-RPMI. Cell suspensions were counted using a Z2 Particle Counter (Beckman Coulter) and viabilities were assessed using 1:4 dilution of cell suspension to 0.2% Trypan Blue under a hemocytometer chamber; mean viability of PBMC was 89.6%. Thawed PBMC were divided for DNA extraction and flow cytometry.
4
Recombinant Human Erythropoietin Evaluation
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Recombinant human EPO (rHuEPO) was purchased from CJ Cheiljedang Corporation (Seoul, Korea). Poly-D-lysine was from Sigma (Saint Louis, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Duchefa Biochemie (Haarlem, the Netherlands). Hanks' balanced salt solution (HBSS), Neurobasal medium, B27 supplement, glutamax I, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid were obtained from GibcoBRL (Grand Island, NY, USA). Dulbecco's modified eagle's medium (DMEM, high glucose-4,500 mg/L, low glucose-1,000 mg/L), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Hyclone Laboratories (Logan, UT, USA). Albumin bovine was obtained from Affymetrix (Santa Clara, CA, USA). Antibodies against phospho-p44/42MAPK (p-ERK1/2), p44/42MAPK (ERK1/2), phospho-p38 MAPK (p-p38 MAPK), p38 MAPK, phospho-SAPK/JNK (p-SAPK/JNK), SAPK/JNK were all purchased from Cell Signaling Technologies (Beverly, MA, USA). Secondary goat anti-rabbit IgG-horseradish peroxidase (HRP) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Clarity Western Enhanced Chemiluminescence (ECL) Substrate was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
5
Cell Culture Conditions for HEK 293T, Huh 7, and Vero E6
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HEK 293 T (American Type Culture Collection (ATCC), CRL-3216), Huh 7 (Japanese Collection of Research Bioresources [JCRB], 0403) and Vero E6 (ATCC, CRL-1586) cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, South Logan, UT). The medium was supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin–streptomycin solution (Gibco, Carlsbad, CA, USA), 2% 4-(2-hydroxy ethyl)-1-piperazineethanesulfonic acid (Gibco, Carlsbad, CA, USA) at 37 °C with 5% CO2.
6
Isolation of Hepatocytes and NPCs
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Two‐month‐old C57bl6 male mice were anesthetized with Nembutal, and an incision was made to open the peritoneum. A 24‐gauge catheter was inserted into the inferior vena cava and another in the portal vein; the inferior vena cava below the heart was clamped. Through the portal vein catheter, the liver was washed with 100 mL calcium (–) magnesium (–) Hank's balanced salt solution (HBSS) with 50 mM 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid (Gibco) and 25 mM ethylene glycol tetraacetic acid at 2‐3 mL/minute. Next, the liver was digested with 0.3 mg/g collagenase type IV (Sigma) and 50 mM 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid in HBSS with calcium. Upon digestion, liver was removed and dispersed in 30 mL HBSS. Dispersed cells were filtered by mesh (100 μm) and centrifuged at 50g for 5 minutes twice to isolate hepatocytes. Then, supernatant was spun down at 700 g for 10 minutes to obtain nonparenchymal cells (NPCs).
7
Exendin-4 Stimulation of Adipose Tissue
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AT was collected in Hank's Balanced Salt Solution (PAA laboratories, Pasching, Austria) supplemented with 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco, Life Technologies, Paisley, UK), 0.1% Gentamicin, 1% Amphotericin (both from Sigma-Aldrich, St Louis, MO, USA). Tissue was dissected into 5–10 mg pieces, removing blood vessels and connective tissue. Approximately 250 mg of tissue was cultured in Media 199 (Gibco, Life Technologies), supplemented with 5% fetal calf serum and 1% penicillin/streptomycin) with increasing concentrations of Exendin-4 (Isca Biochemicals, Exeter, UK), a dipeptidyl peptidase-4-resistant GLP-1R agonist sharing 53% structural homology with GLP-1.8 (link) Tissue explants were incubated at 37 °C for 45 h in an atmosphere of 5% CO2. Media was used for analysis of protein secretion by ELISA and tissue frozen in Tri Reagent solution (Ambion, Life Technologies) for further RNA processing.
8
Culturing HEK293T and K-562 Cells
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HEK293T (American Type Culture Collection [ATCC], CRL-3216) was grown in Dulbecco's modified Eagle's medium (HyClone, South Logan, UT, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin-streptomycin solution (Gibco) and 2% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Gibco) at 37 °C under 5% CO2. K-562 cells (ATCC, CCL-243) were grown in RPMI 1640 (HyClone) supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin solution.
9
Cytotoxicity Evaluation of Plant Extracts
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RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, amphotericin B, 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid, and phosphate-buffered saline (PBS) pH 7.4 were purchased from Gibco® (USA). Ethanol, mEthanol, Giemsa, barium chloride (BaCl2), Folin-Ciocalteu, 1,1- diphenyl-2-picryl hydrazyl (DPPH), cetylpyridinium chloride, sodium acetate, cysteine, ethylenediaminetetraacetic acid (EDTA), crude papain, sodium chloride, sodium carbonate, potassium sulfate, and latex beads were obtained from Sigma-Aldrich (USA).
10
Cell Culture Protocol for Various Cell Lines
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HEK293T (American Type Culture Collection [ATCC], CRL-3216), HEK293FT (Invitrogen, Carlsbad, CA, USA), HEK293 (ATCC, CRL-1573), HepG2 (ATCC, HB-8065),Vero (ATCC,CCL81),Vero E6 (ATCC, CRL-1586), A549 (ATCC, CCL-185), HeLa (ATCC, CCL-2), and BHK21 cells (ATCC, CCL-10) were grown in Dulbecco’s modified Eagle’s medium (HyClone, South Logan, UT, USA) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin–streptomycin solution (Gibco) and 2% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco) at 37 °C under 5% CO2. K-562 cells (ATCC, CCL-243) were grown in RPMI 1640 (HyClone) supplemented with 10% foetal bovine serum and 1% penicillin–streptomycin solution.
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