Dansyl chloride

Polyamines were separated by thin-layer chromatography as previously described [32 ]. For all samples, cells were treated as described prior to being trypsinized and centrifuged. Pellets were washed with PBS and then resuspended in 200 μL 2% perchloric acid. Samples were then incubated overnight at 4 °C. Supernatant (200 μL) was combined with 200 μL 5 mg/mL dansyl chloride (Sigma Aldrich) in acetone and 100 μL saturated sodium bicarbonate. Samples were incubated in the dark overnight at room temperature. Excess dansyl chloride was cleared by incubating the reaction with 100 μL 150 mg/mL proline (Sigma Aldrich). Dansylated polyamines were extracted with 50 μL toluene (Sigma Aldrich) and centrifuged. Five microliters of sample was added in small spots to the TLC plate (silica gel matrix; Sigma Aldrich) and exposed to ascending chromatography with 1:1 cyclohexane/ethyl acetate. The plate was dried and visualized via exposure to UV.

Polyamines were separated by thin-layer chromatography as previously described^{73 (link),74} . For all samples, cells were collected and centrifuged. Pellets were washed with Phosphate Buffered Saline (PBS) and then resuspended in 100 µL of 2% perchloric acid. Samples were then incubated overnight at 4 °C. One hundred µL of supernatant was combined with 200 µL of 5 mg/ml dansyl chloride (Sigma Aldrich) in acetone and 100 µL of saturated sodium bicarbonate. Samples were incubated in the dark overnight at room temperature (RT). Excess dansyl chloride was cleared by incubating the reaction with 100 µL 150 mg/mL proline (Sigma Aldrich). Dansylated polyamines were extracted with 50 µL toluene (Sigma Aldrich) and centrifuged. Five µL of sample was added in small spots to the TLC plate (silica gel matrix; Sigma Aldrich) and exposed to ascending chromatography with 1:1 cyclohexane: ethylacetate. Plate was dried and visualized via exposure to UV.

Derivatization of BA was carried out according to the procedures developed by Ben-Gigirey et al. [24 (link)]. One milliliter of extract (or standard solution) prepared above was mixed with 200 μL of 2 M sodium hydroxide and 300 μL of saturated sodium bicarbonate (all from Sigma-Aldrich). Two milliliters of a dansyl chloride (Sigma-Aldrich) solution (10 mg/mL) prepared in acetone were added to the mixture, and reacted at 40 °C for 45 min. Residual dansyl chloride was removed by adding 100 μL of 25% ammonium hydroxide (Sigma-Aldrich). After reaction at 25 °C for 30 min, the final volume was adjusted to 5 mL with acetonitrile. Finally, the mixture was centrifuged at 3000× g for 5 min, and the supernatant was filtered through a 0.2 μm-pore-size filter (Millipore). The filtered supernatant was kept at −25 °C until assayed by HPLC.

Polyamines were separated by thin-layer chromatography as previously described (23 (link)). For all samples, cells were trypsinized (Zymo Research), reseeded with new medium supplemented with 2% serum, collected, and centrifuged. The pellets were washed with PBS and resuspended in 100 μL of 2% perchloric acid. Samples were then incubated overnight at 4°C. A volume of 100 μL of the supernatant was combined with 200 μL of 5 mg/mL dansyl chloride (Sigma-Aldrich) in acetone (Sigma-Aldrich) and 100 μL of saturated sodium bicarbonate (Sigma-Aldrich). Samples were incubated in the dark overnight at room temperature. Excess dansyl chloride was cleared by incubating the reaction with 100 μL of 150 mg/mL proline (Sigma-Aldrich). Dansylated polyamines were extracted with 50 μL of toluene (Sigma-Aldrich) and centrifuged. A volume of 5 μL of the sample was added in small spots to the TLC plate (silica gel matrix; Sigma-Aldrich) and exposed to ascending chromatography with 1:1 cyclohexane: ethyl acetate. The plate was dried and visualized via exposure to UV. Images were quantified by ImageJ.

The determination of polyamines uses TLC [28 (link)]. For all samples, the cells were washed twice, then the cells were scraped off and centrifuged. The pellet was washed with PBS, and the supernatant removed. A total of 200 μL 2% perchloric acid was added to each cell pellet sample. After these samples were incubated overnight at 4 °C, 200 μL 5 mg/mL dansyl chloride (Sigma-Aldrich, Shanghai, China) acetone and 100 μL saturated sodium bicarbonate were added to each 200 μL supernatant or standard sample (spermine, spermidine, and putrescine) to dansylate the polyamines and neutralize the acid. After incubating these samples overnight in the dark at room temperature, 100 μL of 150 mg/mL proline (Sigma-Aldrich) was added to remove excess dansyl chloride. A total of 500 μL of toluene was used to extract the dansylated polyamine. The hair cell polyamine sample was added to the TLC plate (Dingguo, Beijing, China) at small points, and the solution was chromatographed with a 2:3 cyclohexane–ethyl acetate developing solution for 1.5 h. After the TCL plate was dried, it was visually observed by ultraviolet rays with a wavelength of 365 nm, and the TCL plate image was photographed and stored.

Levels of 2-AAA in cell supernatant were quantified by liquid chromatography mass spectrometry (LCMS) at the Vanderbilt Mass Spectrometry Core. Samples were spiked with internal standard (Arginine-¹⁵N₄, Sigma Aldrich), extracted with methanol, and derivatized with dansyl chloride (Sigma Aldrich) prior to analysis. The dansyl derivative of 2-AAA ([M+H]⁺ 395.1271) was measured by targeted selected ion monitoring (SIM) using a Vanquish ultrahigh performance liquid chromatography (UHPLC) system interfaced to a QExactive HF quadrupole/orbitrap mass spectrometer (Thermo Fisher Scientific). Data acquisition and quantitative spectral analysis were conducted using Thermo-Finnigan Xcaliber version 4.1 and Thermo-Finnigan LCQuan version 2.7, respectively. Calibration curves were constructed by plotting peak area ratios (2-AAA_/Arg-¹⁵N₄) against analyte concentrations for a series of 2-AAA standards. Electrospray ionization source parameters were tuned and optimized using an authentic 2-AAA reference standard (Sigma Aldrich) derivatized with dansyl chloride and desalted by solid phase extraction prior to direct liquid infusion.