Cellmask green actin tracking stain

Late feeding L3 larvae were gently removed from inside the food media using a paintbrush and rinsed in water to remove food particles. Larval fat bodies (FBs) or guts were dissected in PBS using Dumont #5 forceps (Electron Microscopy Sciences). All tissues were fixed in 4% paraformaldehyde for 20 min at room temperature and rinsed briefly in PBS prior to staining with organelle labeling dyes. Lipid droplets in FBs were stained by incubating FBs in 100 µM monodansylpentane (MDH) LD stain (Abgent) for 30 min at room temperature, in the dark. F-actin structures were stained with Phalloidin (1:400) or Cell Mask Green Actin Tracking Stain (1:1000) according to manufacturer’s instructions (Invitrogen). LDs in guts were stained with 1 µM Nile Red for 30 min at room temperature, in the dark. Next, FBs and guts were rinsed in PBS and mounted on slides in SlowFade Gold antifade reagent with DAPI (Invitrogen) for subsequent imaging.

Hep G2 cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/mL. After being cultured for 24 h, Cy3-ANG (0.2 mg/mL as equivalents) in FBS-free culture media was added into cells and incubated for 2 h. Cells were collected after trypsinization and the binding of ANG was detected by flow cytometry using the Cy3 fluorescence. For confocal laser scanning microscopy observation of nanogels internalization, Hep G2 cells were incubated with the Cy3-ANG for 2 h and were fixed in 4% formaldehyde for 15 min at room temperature. The polymerized/filamentous actin in cells was stained by CellMask Green Actin Tracking Stain (Invitrogen) at room temperature for 15 min, while the nucleus was stained by Hoechst (Invitrogen) at room temperature for 25 min. Finally, the samples were detected by confocal laser scanning microscope CrestV2 with 2xTIRF (Nikon).

Following AFM measurements, the cell lines were labeled with F-actin and β-tubulin. Briefly, the cells were fixed for 10 min with 4% (v/v) paraformaldehyde (PFA, Sigma-Adrich) in PBS at room temperature and then washed three times with PBS. For F-actin, a solution of 1μM CellMask™ Green Actin Tracking Stain (#A57243; Thermo Scientific, Waltham, Massachusetts, USA) in 1% (w/v) bovine serum albumin (BSA) in PBS was used for 1 h. For β-tubulin, the cells were then incubated with anti- β-tubulin (rabbit, 1:100, #2129 9F3, Cell Signaling, Danvers, MA, United States) in 1% (w/v) BSA-PBS overnight at 4 °C in a humidity chamber. Afterward, the cells were washed three times with PBS and incubated with a secondary conjugated antibody (Alexa Fluor-594 goat anti-rabbit IgG, #a21429, Thermo Scientific) at a dilution of 1:200 in 1% (w/v) BSA-PBS for 2 h at room temperature in the dark. Nuclear staining was performed with 2 μg/ml DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific). The cells were washed three times for 5 min each in PBS. Images were acquired using a Leica DMi8 microscope (Leica, Wetzlar, Germany) at a 40x objective.