Splenocytes harvested from OT-I mice were stimulated with 10 nM OVA peptide (SIINFEKL) in cRPMI for 24 h. Stimulated T cells were treated with 4 μM of (Z)-4-OHT (Cat #H7904; Sigma-Aldrich), and were cultured for two more days at a cell concentration of 0.2–0.5 × 106 cells/ml in cRPMI supplemented with 10 U/ml rhIL-2 (Cat#202-IL; R&D Systems) in T75 flasks.
Z 4 oht
Manufactured by Merck Group
(Z)-4-OHT is a selective estrogen receptor modulator (SERM) that functions as a tool compound for research purposes. It is commonly used in cell and molecular biology studies to induce gene expression. The compound acts by binding to and activating the estrogen receptor, which can then regulate the transcription of target genes.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rhil 2, by R&D Systems (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Its liquid media supplement, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Win55 212 2, by Bio-Techne (1 mentions)
Cp55 940, by Bio-Techne (1 mentions)
35s gtpγs, by American Radiolabeled Chemicals (1 mentions)
Damgo, by Bio-Techne (1 mentions)
5 protocols using z 4 oht
1
OT-I Splenocyte Stimulation and Expansion
2
Quiescent MEF Activation by MycER and CDK9 Inhibition
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MEFs were generated from embryos 13.5 d.p.f. and cultured in DMEM (Thermo Fisher 41966052) supplemented with penicillin–streptomycin (Thermo Fisher, 15140-122), L-glutamine (Thermo Fisher, 25030-024) and 10% BGS (Hyclone SH30541.03HI). All experiments were performed between passage 3 and 5. For experiments, MEFs were plated at 5 × 105 cells per 6 cm culture dish in 10% BGS (where appropriate, plus adenovirus). To render cells quiescent, the next day cells were washed twice with PBS and cultured in media containing 0.1% BGS for 48 h. After 48 h serum deprivation, cells were treated with 100 nM (Z)-4-OHT (Sigma, H7904) in ethanol to activate MycERT2 and 1 µM AZ5576 (AstraZeneca) in DMSO to block CDK9 and RNA, or protein harvested 4 h later or cell cycle analysis performed after 16 h.
3
Lineage Tracing of Macrophages
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Tg(mfap4:CreER) transgenic fish were outcrossed with Tg(mpeg1:LRLG) reporter line to generate Tg(mfap4:CreER;mpeg1:LRLG) double transgenic fish. After de-chorion, the Tg(mfap4:CreER;mpeg1:LRLG) embryos were incubated with 5 μM (Z)–4-OHT (H7904, Sigma-Aldrich) from 18 to 23 hpf, washed intensively with fresh egg water for at least five times, and changed to a new dish for further incubation.
4
Isolation and Culture of Adult Mouse Cardiomyocytes
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Cardiomyocytes CMs were isolated from adult mouse hearts (8–10 weeks of age) using a simplified Langendorff-free method78 (link) and cultured in M199 (Sigma, M4530) supplemented with 0.1% BSA (supplier, number), 1X ITS Liquid Media Supplement (Sigma, I3146), 10 mmol/l BDM (Sigma, B0753), 1X Chemically Defined Lipid Concentrate (Fisher, 11548846) and penicillin–streptomycin (Thermo Fisher, 15140-122). When needed, adenovirus was administered to CMs in culture media immediately after removal of plating media. Medium was then replaced after 24 h with culture media together with 100 nM (Z)-4-OHT (Sigma, H7904) in ethanol and collected after 4 h for RNA extraction.
5
Ligand Binding Assay for Cannabinoid Receptors
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The following SERMs were purchased from the commercial sources as indicated: E-Tam from Carbosynth (San Diego, CA), Z-Tam from Cayman Chemical (Ann Arbor, MI), E-4OHT from Tocris Bioscience (Ellisville, MO), Z-4OHT from Sigma-Adrich (St. Louis, MO), E-End from Santa Cruz Biotechnology, Inc. (Dallas, TX), and Z-End from Axon Medchem (Reston, VA). WIN-55,212–2, CP-55,940, and DAMGO were obtained from Tocris Bioscience. GTPγS was procured from EMD Chemical (Gibbstown, NJ). [3H]CP-55,940 (131.4 Ci/mmol) was purchased from PerkinElmer (Waltham, MA) and [35S]GTPγS (1250 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Pertussis toxin was acquired from List Biological Laboratories Inc. (Campbell, CA). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA).
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