Tritc phalloidin

Based on a previous study [34 (link)], in order to identify the transfer of lncRNA SNHG12 in EVs, Cy3-labeled SNHG12 was transfected into CAFs. Subsequently, the CAFs expressing Cy3-SNHG12 were seeded into the upper chamber of Transwell 24-well plates, while NSCLC cells were seeded into the lower chamber of Transwell plates for co-culture of 48 h. In addition, the NSCLC cytoskeletons were selectively stained with TRITC Phalloidin (YEASEN, Shanghai, China) or FITC Phalloidin (YEASEN, Shanghai, China). The results were observed with the help of a confocal microscope (Leica Microsystems, Mannheim, Germany).

RAW264.7, EA.hy926, and L929 cells were used to evaluate in vitro cytotoxicity. M@M-Ag-Sil-MA and the three cells were co-cultured in 6-well plates at 37 ℃, after which M@M-Ag-Sil-MA and the medium were removed and replaced with DMEM containing 10% Cell Counting Kit-8 reagent (Biosharp). After incubating for 2 h, the absorbance was measured at 450 nm using a microplate reader (Epoch; BioTEK, Winooski, VT, USA).
Additionally, RAW264.7 cells were stained using the LIVE/DEAD cell imaging kit (Invitrogen, Carlsbad, CA, USA) for 15 min, and EA.hy926 cells were subsequently stained with 100 nM TRITC-phalloidin (Yeasen, Shanghai, China) and 4′,6-diamidino-2-phenylindole (DAPI, Biosharp) to observe cell morphology. Stained cells were observed under an inverted fluorescence microscope (ECLIPSE Ts2; Nikon, Tokyo, Japan).

Cells were seeded in Bio-Oss^® + Gel or Bio-Oss^® as previously described and induced in an osteogenic medium. On day 5, cells were gently washed twice with PBS and fixed with 4% paraformaldehyde solution for 20 min, followed by washing with PBS. Then, 0.3% (v/v) Triton X-100 was applied for 5 min to permeabilize the cell membrane. After three further washes, non-specific binding was blocked with 2% bovine serum albumin for 30 min, and then the cells were incubated with primary antibodies (anti-Runx 2, 1:200, ABclonal, Shanghai China; anti-OCN and anti-MMR, Abcam, Cambridge, UK, 1:200; and anti-iNOS, Santa, 1:200) at 4°C overnight. Cell preparations were then washed with PBS and incubated with the corresponding secondary antibody (Yeason, Shanghai, China, 1:200) for 2 h at room temperature. After three washes, the preparations were incubated in TRITC-phalloidin (Yeason, Shanghai, China) for 30 min, nuclei were counterstained with DAPI, and the cells were washed thoroughly with PBS before qualitative analysis by laser scanning confocal microscopy (Leica, Weztlar, German).

SK-N-SH cells infected with or without HSV-1 were treated with amentoflavone (50 μM) or cytochalasin B (CB, 20 μM). After 1 h at 37 °C, the cells were washed with PBS, fixed with 4% paraformaldehyde for 5 min, and permeabilized with 0.1% Triton X-100 for 5 min. Next, the samples were stained with 5% TRITC-Phalloidin (YEASEN, 40734ES75) for 40 min at 37 °C. Finally, the fluorescence was analyzed with a flow cytometer (Becton Dickinson, CA, USA).

The fixed embryos were washed three times with PBS containing 0.1% Triton X-100 (0.1% PBST). Then, the fixed embryos were stained with phalloidin for 12 h at 4 ºC, finally washed three times with PBS containing 0.1% Triton X-100 (0.1% PBST). Alexa Fluor 488 (Invitrogen) was dissolved with 1.5 ml of methanol and diluted (1:300) to mark the cell boundary of embryos. TRITC phalloidin (YEASEN) also was diluted (1:300) to mark the cell boundary. DAPI was used in the dark to mark the cell nucleus. Embryos were imaged using a Nikon A1 confocal microscope and a Zeiss LSM900 confocal microscope.