1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii dye

TIPP (AEWCP, purity ≥ 99%, molecular weight 604.68 Da), IS (S4: AEWSP, purity ≥ 99%, molecular weight 588.61 Da), and FITC-TIPP (5-FITC-(Ahx)-AEWCP (Figure 1c), purity ≥ 96%, molecular weight 1107.19 Da) were synthesized by QIANGYAO Biological Technology Company (Wuhan, China). ACN, FA, DCM, isopropanol (IPA), and MeOH, of LC-MS grade, were from Merck Darmstadt, Germany. DTT, L-AA, PMSF, monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype, clone SPE-7 (anti-DNP-IgE), dinitrophenyl-human serum albumin (DNP-HSA), 1,4-piperazinebis (ethanesulfonic acid) (PIPES), p-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) dye, and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Minimum Essential Medium (MEM) and fetal bovine serum (FBS) were obtained from Gibco (Waltham, MA, USA). TDW was obtained from Milli-Q PLUS PF water purifying system (Millipore, Bedford, MA, USA). Rat IL-4 and histamine ELISA detection kits were produced by Elabscience Biotechnology Company (Wuhan, China).

HEK293 or C6 cells were incubated with pDNA-GVs (pDNA-GVs@ HEK293 cells or pDNA-GVs@ C6 cells) for 2, 4, or 6 h at 37 °C, and free pDNA-GVs was removed via PBS rinse. To evaluate the cellular uptake efficiency of pDNA-GVs, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (Dii) dye (Sigma-Aldrich)-labeled pDNA-GVs were incubated with HEK293 or C6 cells, and DAPI (Sigma-Aldrich) was used to stain the pDNA-GVs@HEK293 cells or pDNA-GVs@C6 cells, followed by assay with a confocal microscope (A1, Nikon, Japan) and flow cytometry (CytoFLEX LX, Beckman, USA). TEM was used to examine the morphology and subcellular localization of pDNA-GVs in HEK293 and C6 cells (HT7700, HITACHI, England). To locate pDNA-GVs in lysosomes, pDNA was labeled with N,N’-(dipropyl)-tetramethyl indocarbocyanine (Cy3) using Label IT Nucleic Acid Labeling Kit (Mirus) and following supplier’s protocol. The fluorescent labeling pDNA/PEI_{25 k} or pDNA-GVs complexes were used to incubate with HEK293 cells or C6 cells for 6 h. After that, the cells were washed with PBS, and stained with LysoTracker Green (Thermo Fisher) for lysosome and DAPI (Sigma-Aldrich) for nuclei. Finally, the cells were observed under a confocal microscope (A1, Nikon, Japan).