Mitotracker red

The subcellular localization of SSSe NPs was investigated by confocal microscopy. Briefly, 1 × 10⁴ H9C2 cells were plated on poly‐L‐lysine‐treated coverslips in 24‐well plates and incubated with or without 0.7 µg ml⁻¹ SSSe NPs‐FITC for 4 h. Then, the cells were stained with MitoTracker Red (Yeasen, Shanghai, China) at 37 °C for 2 h. After being washed, the cells were stained with Hoechst at 37 °C for 10 min. A confocal laser scanning microscope (Zeiss, LSM900, Germany) was used for cellular imaging. The images were analyzed by using the Just Another Colocalization Plugin (JACoP) and ImageJ software (NIH, MD, US).

The full-length coding sequence of ZmPLD3 without the stop codon was cloned into pCAMBIA1300-35S::eGFP for subcellular localization analysis. The resulting pCAMBIA1300-35S::ZmPLD3-eGFP vector was subsequently cotransformed with AtHDEL-mCherry (an endoplasmic reticulum marker)^{39 (link)}, AtSYP61-mCherry (a Golgi marker)^{40 (link)}, WxTP-mCherry (a plastid marker)^{41 (link)}, AtCBL1-mCherry (a plasma membrane marker)^{42 (link)}, AtAHL22-mCherry (a nuclear marker)^{43 (link)}, AtVSR2-mCherry (a prevacuolar compartment marker)^{44 (link)}, AtPTS1-mCherry (a peroxisome marker)^{45 (link)} or free mCherry (a cytosol marker)^{46 (link),47 (link)} into maize protoplasts. A pCAMBIA1300-35S::eGFP unmodified vector was also cotransformed with the same markers into maize protoplasts, which served as controls. After culturing at 28 °C for 18 h, fluorescent signals were detected using a confocal microscope (Zeiss 880). For observation of mitochondrial localization, only pCAMBIA1300-35S::ZmPLD3-eGFP vector or pCAMBIA1300-35S::eGFP unmodified vector was transformed into maize protoplasts and after culturing at 28 °C for 18 h, protoplasts were incubated with the diluted mitochondrial red fluorescent probe MitoTracker Red (25 nM) (40741ES50; YEASEN) for ~30 min and then detected with confocal microscope.

HeLa cells (1 × 10⁵) were seeded in six-well plates and treated with FITC-labeled PEGcleavable Tf-CTM/L for 2 h after reaching 80% of the overspread. The concentration of various formulations was calculated as FITC (10 µM). After incubation, HeLa cells were rinsed with PBS three times, and then collected by trypsin without EDTA to harvest in 0.2 mL of PBS via flow cytometry (Guava 6HT, Merck Millipore).
According to previous studies, the intracellular delivery of various tripterine treatments was studied (Qu et al., 2015 , 2017 (link); Chen et al., 2018 ; Qu et al., 2018 (link)). HeLa cells (1 × 10⁵) were seeded in 12-well plates and treated with FITC-labeled PEGcleavable Tf-CTM/L at a FITC concentration of 10 µM. After 2 h of incubation, HeLa cells were washed by PBS (thrice, ice cold), followed by staining with MitoTracker Red (100 nM, Yeasen, China) and LysoTracker Red (50 nM, Abcam, UK). Finally, HeLa cells were fixed via 4% (v%) paraformaldehyde (25 °C) and observed by CLSM.