9 aminoacridine 9 aa

The standard of forchlorfenuron (purity > 98%) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). Forchlorfenuron-d5, the isotopic internal standard (IS), was purchased from Toronto Research Chemicals (Toronto, ON, Canada). HPLC-grade methanol, formic acid, and pure water were obtained from Fisher (Marshalltown, IA, USA). MALDI-grade α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 9-aminoacridine (9-AA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other reagents and solvents used in the present study were of analytical grade.

The isoflurane anaesthetic and nitrogen 4.0 were obtained from Baxter and Linde (both Bratislava, Slovak Republic), respectively. The indium-tin oxide (ITO) glass slides and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Bruker Daltonics (Bremen, Germany). Acetonitrile, methanol, ethanol and water (all LC-MS grade) were obtained from Merck Millipore (Prague, Czech Republic). Trifluoroacetic acid (TFA), chloroform, sodium chloride, 9-aminoacridine (9-AA), haematoxylin solution, Gill no. 2 and DPX mounting media were obtained from Sigma-Aldrich (Prague, Czech Republic). Eosin Y (0.5%) aqueous solution was purchased from VWR Chemicals (Stribrna Skalice, Czech Republic). Hydrochloric and nitric acid (65%, Analpure) were obtained from FlukaBioChemika or Analytika Ltd., respectively (both Prague, Czech Republic). Deionized water (18.2 MΩ/cm) was prepared using a Milli-Q system (Millipore, Molsheim, France). Omni Slides with hydrophobic surfaces were purchased from Prosolia, Inc. (Zionsville, USA).

9-aminoacridine (9AA) was from Sigma-Aldrich (St. Louis, MO). For in vitro studies, stock solutions of 9AA were made in dimethylsulfoxide (DMSO), and subsequently diluted in RPMI 1640 medium for use. For in vivo studies, 9AA was dissolved in a solution with 10% polyethylene glycol 300 (PEG300) (VWR, Bridgeport, NJ) and continuously administrated via a subcutaneous mini-osmotic pump (Alzet, Cupertino, CA). Campath-1H was from Genzyme Corporation (Cambridge, MA) and diluted in PBS before use.

Inositol phosphates from D. discoideum cell extract were purified as described above and previously [28] (link) and directly subjected to mass spectrometry [37] (link). Matrix-assisted laser desorption ionization (MALDI) mass spectrometry was performed on a Voyager DE-STR (Applied Biosystems, Framingham, MA), equipped with a MALDI ion source and a time-of-flight mass analyzer (MALDI-TOF). 9-aminoacridine (9-AA, Sigma-Aldrich) was used as matrix, due to its superior performance in revealing acidic analytes in negative ion mode [37] (link). A double deposition sample preparation procedure was adopted. Typically, 0.5 µL of matrix solution, consisting of 7 mg/mL 9-AA in a 1∶1 mixture (v/v) of acetonitrile and water was spotted on the stainless steel MALDI sample stage and air-dried. Then, 0.5 µL of the analyte solution, either pure or diluted 1∶5 (v/v) in water, was spotted on to the matrix crystals and allowed to dry. Mass spectra were acquired in delayed extraction, reflectron negative ion mode using the following settings: accelerating voltage 20,000 V, grid voltage 73%, extraction delay time 300 nsec, acquisition mass range 300–1,500 m/z. Each spectrum was the average of 400–500 individual laser shots acquired in series of 100 consecutive shots.