Splenocytes were isolated and cultured in IL-15 (15 ng/ml; PeproTech) at 37°C for 6 days. On day 4, the cells were supplemented with IL-15 (15 ng/ml) and cultured for an additional 2 d. On day 6, cultured NK cells were magnetically purified (NK cell Isolation Kit II, Miltenyi) and stimulated for 18 hr with IL-2 (20 ng/ml; National Cancer Institute Preclinical Repository) and/or IL-12 (10 ng/ml; Miltenyi Biotec) cytokines. Low-dose IL-15 (6.66 ng/ml) was added as a survival factor to unstimulated cultures. Experiments were carried out in the presence or absence of TEPP-46 (Cayman Chemical or EMD Millipore), rapamycin (20 nM; Fisher), N-acetyl-cysteine (7.5 mM; EMD Millipore) or dehydroepiandrosterone (DHEA) (75 μM, Sigma Aldrich). Splenocytes were cultured in RPMI medium containing 10% FBS, 2 mM glutamine (Thermo Fisher), 50 μM 2-ME (Sigma-Aldrich), and 1% Penicillin/Streptomycin (Thermo Fisher).
Dehydroepiandrosterone dhea
Manufactured by Merck Group
Sourced in United States, Macao, United Kingdom
Dehydroepiandrosterone (DHEA) is a chemical compound that is naturally produced by the adrenal glands in the human body. It is a precursor to other hormones, including testosterone and estrogen. DHEA is often used in research and laboratory settings to study the role of hormones in various biological processes.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Progesterone, by Merck Group (3 mentions)
Testosterone, by Merck Group (3 mentions)
Formic acid, by Merck Group (3 mentions)
Rapamycin, by Thermo Fisher Scientific (2 mentions)
Nk cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Il 12, by Miltenyi Biotec (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions)
Il 15, by Miltenyi Biotec (2 mentions)
Tepp 46, by Cayman Chemical (2 mentions)
Cortisone, by Merck Group (2 mentions)
Estrone, by Merck Group (2 mentions)
Cortisol, by Merck Group (2 mentions)
Dihydrotestosterone dht, by Merck Group (2 mentions)
17β0 estradiol, by Merck Group (2 mentions)
D5 estradiol, by CDN Isotopes (2 mentions)
D5 testosterone, by CDN Isotopes (2 mentions)
D4 dht, by CDN Isotopes (2 mentions)
10 protocols using dehydroepiandrosterone dhea
1
Isolation and Activation of NK Cells
2
Steroid Reference Materials for LC-MS/MS
3
DHEA and Rha Effects on KGN Cells
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We purchased KGN cells from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) including 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% gentamicin, and incubated in a humidified atmosphere condition at 37 ℃ with 5% CO2. Different concentrations of dehydroepiandrosterone (DHEA; 10 nM, 100 nM, 1 µM, 10 µM, and 100 µM, Sigma-Aldrich, St. Louis, MO, USA) were then treated for 48 hours. Cells were incubated with low, medium, or high doses of Rha (Rha; 1 µM, 10 µM or 100 µM; Carbosynth, UK).
4
Quantitative Steroid Analysis Protocol
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Testosterone, androstenedione, DHT, DHA, epiTestosterone (EpiT), dehydroepiandrosterone (DHEA), formic acid (FA) ≥98%, hydrazine monohydrate, dichloromethane (DCM), trifluoroacetic acid (TFA), ammonium acetate and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS) were from Sigma-Aldrich, (Dorset, UK). 2,3,4-13C3-Testosterone (13C3-T), 99%, 2,3,4-13C3-androstenedione (13C3-A4), ≥98%, 2,3,4-13C3-5α-dihydroTestosterone (13C3-DHT), ≥97% were from IsoSciences (Philadelphia, USA). Certified solutions of Testosterone, androstendione (both 1 mg/mL in acetonitrile) and DHT (1 mg/mL in methanol) and 13C3-T and 13C3-A4 (100 µg/mL in acetonitrile) were from Cerilliant (Texas, USA). HPLC grade glass distilled solvents (methanol, acetonitrile, acetone, n-hexane, DCM and water) and LC-MS grade solvents (methanol, acetonitrile and water) were from Fisher Scientific UK Limited (Leicestershire, UK).
5
Steroid Reference Materials for LC-MS/MS
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Steroid reference materials were obtained from Sigma-Aldrich (St. Louis, MO): estrone, 17β0-estradiol, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), progesterone, cortisol and cortisone. The deuterated internal standards, d5-estradiol, d5-testosterone, d4-DHT, d9-progesterone and d4-cortisol were obtained from CDN Isotopes (Point-Claire, Quebec, Canada). HPLC grade methanol, 2-propanol, water, and formic acid were purchased from Sigma.
6
Baicalin and DHEA Signaling Pathway
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Baicalin and dehydroepiandrosterone (DHEA) were purchased from Sigma-Aldrich, USA. Antibodies against AMPK, P-AMPK, Akt, and P-Akt were acquired from Cell Signaling Technology Inc., USA. Antibodies against 5α-R1 and β-actin from Santa Cruz Biotechnology, USA. RIPA lysis from Beotime Corporation, Beijing, China. BCA TM protein assay kit from Pierce Chemical Company, Rockford, USA.
7
Mammary Epithelial Cell Culture
8
Microbial Transformation of DHEA
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The substrates androstenedione, adrenosterone, progesterone, 17α-methyltestosterone and dehydroepiandrosterone (DHEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3β,7β-Dihydroxyandrost-5-ene-17-one (7β-OH-DHEA), 3β,7β-dihydroxyandrost-5-ene-17-one (7β-OH-DHEA) and 3β-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) were isolated as a products of DHEA transformation in the culture of Isaria fumosorosea KCh J2 strain.
The microorganism Isaria fumosorosea KCh J2 was obtained from the collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences (Wrocław, Poland). Isolation and identification procedures were described in our previous paper [82 (link)]. The strain was maintained on Sabouraud 4% dextrose-agar slopes and freshly subcultured before use in the transformation experiments.
The microorganism Isaria fumosorosea KCh J2 was obtained from the collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences (Wrocław, Poland). Isolation and identification procedures were described in our previous paper [82 (link)]. The strain was maintained on Sabouraud 4% dextrose-agar slopes and freshly subcultured before use in the transformation experiments.
9
Palbociclib Glucuronidation and Sulfation Assay
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ABT (Sigma‐Aldrich), palbociclib sulfate (TRC), uridine 5′‐diphosphoglucuronic acid, UDPGA (Sigma‐Aldrich), MgCl2 (Sigma‐Aldrich), Alamethicin (Sigma‐Aldrich), dimethyl sulfoxide, DMSO (Sigma‐Aldrich), ACN (liquid chromatography‐mass spectrometry [LC–MS] grade; Fisher Chemical), ACN (Optima LC/MS grade, Fisher Chemical) formic acid (Fisher Chemical), potassium phosphate (Sigma‐Aldrich) 5,5‐diethyl‐1,3‐diphenyl‐2‐iminobarbituric acid (“39:an”; Sigma‐Aldrich), D‐Saccharolactone (Sigma‐Aldrich), ultra‐pure water (Millipore), recombinant UGT supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15; Corning), β‐estradiol (Sigma‐Aldrich), chenodeoxycholic acid (CDCA; Sigma‐Aldrich), trifluoperazine (TFP; Sigma‐Aldrich), serotonin (Sigma‐Aldrich), propofol (Sigma‐Aldrich), zidovudine (Sigma‐Aldrich), oxazepam (Sigma‐Aldrich), raloxifene (Sigma‐Aldrich), dehydroepiandrosterone (DHEA; Sigma‐Aldrich), DHEA‐sulfate (Sigma‐Aldrich), 4‐methylumbelliferone (4‐MU; Sigma‐Aldrich), palbociclib sulfate (Toronto Research Chemicals); recombinant sulfotransferases (SULT1A1, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, SULT2A1, and SULT2B1; R&D Systems), palbociclib, and ARV‐471 were synthesized by AZ chemistry.
10
Cytokine-Induced NK Cell Activation
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Splenocytes were isolated and cultured in IL-15 (15 ng/ml; PeproTech) at 37°C for 6 d. On day 4, the cells were supplemented with IL-15 (15 ng/ml) and cultured for an additional 2 d. On day 6, cultured NK cells were magnetically purified (NK cell Isolation Kit II, Miltenyi) and stimulated for 18 h with IL-2 (20 ng/ml; National Cancer Institute Preclinical Repository) and/or IL-12 (10 ng/ml; Miltenyi Biotec) cytokines. Low-dose IL-15 (6.66 ng/ml) was added as a survival factor to unstimulated cultures.
Experiments were carried out in the presence or absence of TEPP-46 (Cayman Chemical or EMD Millipore), rapamycin (20 nM; Fisher) or dehydroepiandrosterone (DHEA) (75 µM, Sigma Aldrich). Splenocytes were cultured in RPMI medium containing 10% FBS, 2 mM glutamine (Thermo Fisher), 50 μM 2-ME (Sigma-Aldrich), and 1% Penicillin/Streptomycin (Thermo Fisher).
Experiments were carried out in the presence or absence of TEPP-46 (Cayman Chemical or EMD Millipore), rapamycin (20 nM; Fisher) or dehydroepiandrosterone (DHEA) (75 µM, Sigma Aldrich). Splenocytes were cultured in RPMI medium containing 10% FBS, 2 mM glutamine (Thermo Fisher), 50 μM 2-ME (Sigma-Aldrich), and 1% Penicillin/Streptomycin (Thermo Fisher).
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