Label it nucleic acid labeling kit

Cy3-pre-miR-134 and the respective chimeras were in vitro synthesized with the MEGAshortscript T7 kit (Ambion) from oligonucleotide templates and labeled with the Label IT nucleic acid labeling kit (Mirus) as previously described (Bicker et al., 2013 (link)). DIV7 rat hippocampal neurons were transfected with 75 nM Cy3-pre-miRNAs in NB+ without antibiotics using siPORT NeoFX (Ambion) as previously described (Bicker et al., 2013 (link)). Cells were pre-incubated in transfection medium containing 50 μM DL-APV or Mock for 30 min and then transfected and treated simultaneously with 50 ng/ml BDNF or Mock for 3 h. Cells were fixed in 4% paraformaldehyde/4% sucrose/DEPC-PBS and stained with mouse monoclonal α-MAP2 (1:2000, Sigma Aldrich, M9942) and rabbit polyclonal α-cFos (1:1000, Cell Signalling #2250) as previously described (Fiore et al., 2009 (link)).

The decoy ODN was labeled with FITC using a Label IT nucleic acid labeling kit (Mirus Bio). SZ95 cells were cultured in six-well plates and transfected with 2 μg of FITC-labeled decoy ODN or with non-labeled decoy ODN using Lipofectamine 2000. For the flow cytometric analysis, the cells were washed, trypsinized, dispersed, and then transferred into 500 μL PBS. The samples were analyzed with CytExpert (Beckman Coulter, Brea, CA, USA).

β-Glutamic acid hydrochloride and dimethyl sulfoxide (DMSO) were purchased from Fluka (Buchs, Switzerland). L-Histidine dihydrochloride, diethyl ether, DAPI (4′,6-diamidino-2-phenyindole, dilactate) and PBS were purchased from Sigma Co. (St Louis, MO). Poly(ethylene glycol) (PEG) diacrylate (Mn=258, density=1.11 g ml⁻¹) and branched polyethyleneimine (bPEI_25K, Mw=25,000) were purchased form Aldrich (Milwaukee, MI). Plasmid DNA of pCMV vector (pcDNA 3.1) as a null control was purchased from Invitrogen (Carlsbad, CA, USA). Plasmid DNAs of pGL3, pCMV-p53 and pKillerRed-mem were purchased from Promega (Madison, WI, USA), Clontech Laboratories (Mountain View, CA) and Evrogen JSC (Moscow, Russia), respectively. For the preparation of fluorescent-labelled plasmid DNA, pCMV-p53 and pKillerRed-mem stocks were labelled with fluorescein as a fluorescent dye using a Label IT Nucleic Acid Labeling Kit (Mirus Bio, Madison, WI).

pTyr-C9AP was first labeled with Cy5 using a Label IT Nucleic Acid Labeling Kit (Mirus Bio., WI, USA) and then encapsulated to prepare Cy5-labeled NP_Tyr-C9AP. B16-F10, CT26, Panc02, 4T1, C2C12, NIH-3T3, DC2.4 and RAW264.7 cells were seeded in 24-well plates (5 × 10⁴ cells per well) and then incubated with Cy5-labeled NP_Tyr-C9AP at a final concentration of 1 μg ml⁻¹ pTyr-C9AP for 4 h. For CLSM imaging, the cells were fixed with 4% paraformaldehyde, and the F-actin and nucleus were labeled with Alexa Fluor 488 Phalloidin (Invitrogen, MA, USA) and DAPI (Biosharp, Anhui, China) before visualization by an LSM880 (Zeiss, Oberkochen, Germany). The confocal data were collected using ZEISS ZEN2 (black edition) software and analyzed with ZEISS ZEN2 (blue edition) software. For flow cytometry analysis, the cells were collected by trypsinization and detected by FACSCelesta (BD Biosciences, CA, USA). The flow cytometry data were collected using the BD FACS Diva software v8.0.1.1 and further analyzed by FlowJo software v10.0.7 (BD Biosciences).