For vorinostat (Sigma), we chose a range of 50nM– 1 μM. The high dose of 1μM was chosen as it was used in in vitro latency experiments and the peak serum concentration of vorinostat in vivo was reported to be 1.2μM; however as the free concentration of vorinostat was only 360nM [27 (link)], we used 333nM as our physiologically relevant dose. For panobinostat (Cambridge Bioscience), we chose a range from 5-100nM. The mean clinical Cmax of panobinostat at the 20mg p.o. dosing of the CLEAR trial is 40nM [28 (link)]and the steady-state plasma concentration is 15-22nM [7 (link)] so we chose 20nM as our physiological dose. For romidepsin (Abcam), we chose a range of 5-100nM to match our panobinostat range. The peak serum concentration for clinical doses is 698nM with free drug concentration being 56nM [27 (link)]. We thus chose 10nM, which incorporated our cell line toxicity studies. We used 300nM prostratin based on other ex vivo studies [29 (link)].
Romidepsin
Manufactured by Abcam
Sourced in United Kingdom
Romidepsin is a histone deacetylase (HDAC) inhibitor. It is used as a tool compound for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Vorinostat, by Merck Group (3 mentions)
Panobinostat, by Cambridge Bioscience (3 mentions)
Panobinostat, by Abcam (1 mentions)
Sterile dmso, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Abcam (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Abcam (1 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Romidepsin, by Selleck Chemicals (1 mentions)
Methacholine, by Merck Group (1 mentions)
Anti total akt 4691, by Cell Signaling Technology (1 mentions)
Fps zm1, by Selleck Chemicals (1 mentions)
Jnj 26482585, by Selleck Chemicals (1 mentions)
Phospho akt 4060, by Cell Signaling Technology (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Acetone, by Merck Group (1 mentions)
5 protocols using romidepsin
1
Optimization of HDAC Inhibitor Dosing
2
DEN-Induced Hepatocellular Carcinoma in Mice
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C56BL/6 male mice weighing 25–27 gr were purchased from the Hellenic Pasteur Institute. Mice were kept in stainless cages at constant temperature (22 to 24°C) and allowed free access to food and water during the 24-h day/night cycle. All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals.26 Ethical approval and licensing (License reference no 4956/09-08-2012) were provided by the competent National Veterinary Administration Authorities according to Greek legislative (Decree no. 2015/92, 160/91) and European Communities Council directive (no. 86/609/EEC).
A total of 23 male mice were used. Of these, 17 were treated with the carcinogen DEN, and 6 remained untreated. DEN (5 mg/kg of body weight) was given with a single i.p. injection at the age of 14 days for the induction of HCC. At the age of 10 months, 8 of the 17 mice were further i.p. injected with 0.03 mg of the HDAC1/2 inhibitor Romidepsin (Abcam, Cambridge, UK; 0.03 mg/per mouse) twice a week for three consecutive weeks. Romidepsin was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM and further diluted in 500 μL saline prior to i.p. administration. An equal amount of DMSO was diluted in saline and injected i.p. in the remaining 9 mice. Mice were killed with an overdose of ketamine and xylazine at twelve months of age.
A total of 23 male mice were used. Of these, 17 were treated with the carcinogen DEN, and 6 remained untreated. DEN (5 mg/kg of body weight) was given with a single i.p. injection at the age of 14 days for the induction of HCC. At the age of 10 months, 8 of the 17 mice were further i.p. injected with 0.03 mg of the HDAC1/2 inhibitor Romidepsin (Abcam, Cambridge, UK; 0.03 mg/per mouse) twice a week for three consecutive weeks. Romidepsin was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM and further diluted in 500 μL saline prior to i.p. administration. An equal amount of DMSO was diluted in saline and injected i.p. in the remaining 9 mice. Mice were killed with an overdose of ketamine and xylazine at twelve months of age.
3
Epigenetic Modulator Reconstitution Protocol
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Vorinostat was purchased from Sigma-Aldrich (Poole, UK) and panobinostat was ordered from Cambridge Bioscience (Cambridge, UK). They were each reconstituted in sterile DMSO (Sigma-Aldrich) to a concentration of 20 mM. Romidepsin was purchased from Abcam (Cambridge, UK) and reconstituted at a concentration of 2 mM in DMSO. Prostratin was obtained from Cambridge Bioscience.
Phorbol 12-myristate 13-acetate (PMA) was purchased from Abcam and reconstituted with DMSO at a concentration of 1 mg/mL. Recombinant interleukin-1β (IL-1β) was purchased from Life Technologies (Paisley, UK) and reconstituted at a concentration of 1 μg/mL in pure dH2O.
Phorbol 12-myristate 13-acetate (PMA) was purchased from Abcam and reconstituted with DMSO at a concentration of 1 mg/mL. Recombinant interleukin-1β (IL-1β) was purchased from Life Technologies (Paisley, UK) and reconstituted at a concentration of 1 μg/mL in pure dH2O.
4
Isolation and Culture of CD4+ T Cells
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CD4 T cells were negatively selected from PBMC using the EasySEP human CD4 T cell enrichment kit (StemCell Technologies) per the manufacturer’s protocol. Cells were then cultured in RPMI containing 10%FCS, penicillin/streptomycin and L-glutamine (R10). HDACi treated cells were cultured in 1μM or 333nM vorinostat (Sigma), 100nM or 20nM panobinostat (Cambridge Bioscience), or 10nM romidepsin (Abcam).
5
Evaluating RAGE and HDAC Inhibitors in Toluene Diisocyanate Exposure
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TDI (toluene-2, 4-diisocyanate, ≥ 98.0%), acetone, and methacholine were obtained from Sigma-Aldrich (Shanghai, China). The vehicle that TDI was dissolved in was a mixture of acetone and olive oil (AOO): the ratio of acetone to olive oil was 2:3 for sensitization, and 1:4 for challenge. FPS-ZM1 (RAGE inhibitor), JNJ-26482585 (HDAC inhibitor), romidepsin (HDAC inhibitor), and MK2206 (AKT inhibitor) were purchased from Selleck (SelleckChem, Shanghai, China). JNJ-26482585 is a novel second-generation HDACi with highest potency for HDAC1 and romidepsin is a selective inhibitor of HDAC1 and HDAC2.
Anti-RAGE (ab37647), and anti-HDAC1 (ab109411) were from Abcam. Anti-total-AKT (#4691 ) and Phospho-Akt ( #4060 ) were purchased from Cell Signaling Technology (Boston, MA, USA).
Anti-RAGE (ab37647), and anti-HDAC1 (ab109411) were from Abcam. Anti-total-AKT (#4691 ) and Phospho-Akt ( #4060 ) were purchased from Cell Signaling Technology (Boston, MA, USA).
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