3 amino 4 aminomethyl 2 7 difluorescein diacetate daf fm da

1,6-Hexanediamine (92%), N,N-bis(3-aminopropyl)methylamine (98%), chloroform (99%), sodium hydroxide (50 wt% aqueous solution) and benzyltriethylammonium chloride (TEBAC) were purchased from Wanqing Chemical Instrument Co., Ltd (Nanjing, P.R.C.). Paclitaxel, oxaliplatin, cisplatin (DDP), verapamil (VRP) and z-VAD-FMK were obtained from Selleck Chemicals (Houston, USA). Annexin V-FITC/PI double-staining kit was provided by KeyGen (Nanjing, P.R.C.). N-acetyl-L-cysteine (NAC), hemoglobin, Hoechst 33342, crystal violet, 4’,6-diamidino-2-phenylindole (DAPI), 3-amino-4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM DA) and 2’,7’-dichlorofluorescein (DCFH-DA) were provided by Beyotime Biotechnology (Nanjing, P.R.C.). Propidium iodide (PI), calf thymus DNA (ct-DNA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and ethidium bromide (EB) were purchased from Sigma-Aldrich (St. Louis, USA). γ-H2AX (phospho S139) antibody was obtained from Abcam (Cambridge Science Park, Cambridge, UK). Primary antibodies against specific for cleaved caspase-3, cleaved caspase-9, Bax, poly (ADP-ribose) polymerase (PARP), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Boston, USA).

We divided the cultures grown in liquid medium into a T. rubrum group, a T. rubrum+HaCaT cell group, and a T. rubrum+L-NMMA+HaCaT cell group. We pretreated the fungus with L-NMMA for 2 h before coculture with HaCaT cells. A T. rubrum suspension of each group was incubated with 5 μmol/l 3-amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) (Beyotime Biotechnology, Haimen, China), a fluorescent probe, at 37°C for 20 min in a CO₂ incubator and washed in PBS three times. DAF-FM DA can pass through the cell membrane. After entering the cell, it can be catalyzed by intracellular esterase to form DAF-FM, which cannot pass through the cell membrane. DAF-FM itself has only very weak fluorescence but can generate strong fluorescence after reacting with nitric oxide; thus, the degree of NO production can be detected. The fluorescence signal intensity was analyzed using a flow cytometer (BD FACSCanto II) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Based on the fluorescence signal intensity, we could detect the level of NO in T. rubrum.