For non-screen drug plating, 2.5–5×103 live cells were seeded onto flat-bottom 96-well plastic plates in 200 μL of media per well. Drugs were added such that the total DMSO (Sigma-Aldrich) concentration was the same for all wells. After treatment, plates were transferred onto round-bottom 96-well plastic plates (Greiner Bio-One). Cells were analyzed on an iQue Screener Plus-VBR flow cytometer with a similar gating strategy as the one used for the screen, except we isolated singlets using both height and area parameters for FSC and SSC from live cells. We then isolated GFP + or antibody-positive events from the live singlets. GSK-LSD1, mercaptopurine (Tocris), cerulenin (Cayman Chemical), 6-thioguanine (Cayman Chemical), guanosine (Cayman Chemical), TOFA (MedChemExpress), ND-630 (MedChemExpress), hypoxanthine (Sigma Aldrich), and 13C5-hypoxanthine (Cambridge Isotope Laboratories) were dissolved in DMSO as 10mM stocks. Tranylcypromine (Sigma Aldrich) and homocysteine (Sigma Aldrich) were dissolved in ethanol and water respectively as 100mM stock. APC-CD11b antibody (BioLegend) and propidium iodide (ThermoFisher) were used at concentrations of 1 μg/mL or 50 μg/mL respectively.
13c5 hypoxanthine
Manufactured by Cambridge Isotopes
Sourced in Macao
13C5-hypoxanthine is a stable isotope-labeled compound used as an analytical standard or tracer in various research and analytical applications. It contains five carbon-13 atoms, which can be useful for studying biochemical processes or quantifying compound levels using techniques like mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Hypoxanthine, by Merck Group (2 mentions)
Tranylcypromine, by Merck Group (1 mentions)
Homocysteine, by Merck Group (1 mentions)
Cerulenin, by Cayman Chemical (1 mentions)
Apc cd11b antibody, by BioLegend (1 mentions)
6 thioguanine, by Cayman Chemical (1 mentions)
Gsk lsd1, by Bio-Techne (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
8 aminoguanine, by LGC (1 mentions)
Inosine, by Merck Group (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
Guanosine, by Merck Group (1 mentions)
Uric acid, by Merck Group (1 mentions)
2 protocols using 13c5 hypoxanthine
1
Non-Adherent Cell Drug Screening
2
Purine Nucleoside Phosphorylase Assay
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8-Aminoguanine was from Toronto Research Chemicals (Toronto, Ontario, Canada); 8-aminoinosine and 8-aminohypoxanthine were from Santa Cruz Biotechnology, Inc. (Dallas, TX); recombinant human purine nucleoside phosphorylase (rhPNPase) was from R&D Systems (Minneapolis, MN); xanthine oxidase, inosine, hypoxanthine, guanosine, guanine, and uric acid were from MilliporeSigma (St. Louis, MO); 15N4-inosine and 13C5-hypoxanthine were from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA); and 13C10,15N5-guanosine; 13C2,15N-guanine were from Medical Isotopes, Inc. (Pelham, NH).
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