Lsm 510 meta

The endocytosis of the B16F10 cells on the PCP-Mn-DTA nanosystem was observed by CLSM and quantitatively analyzed by FCM. On one hand, the B16F10 cells seeded on the confocal microscope dish were treated with PBS, FITC-labelled GOx (GOx-FITC, 10 mU/mL) and PCP-Mn-DTA@GOx-FITC@1-MT at pH 7.4 and 6.8 for 4 and 12 h. The cells were subsequently fixed with 4% paraformaldehyde, permeabilized with 0.5% TritonX-100, followed by staining with ActinRed 555 and 4’,6-diamidino-2-phenylindole (DAPI). CLSM (LSM 510 META Olympus) and CellSens Dimension software were employed to study the efficiency of uptake by the B16F10 cells. On the other hand, the B16F10 cells treated with the various systems were washed with PBS. The cells were subsequently centrifuged (800 g × 10 min) at 4 °C and collected. Finally, they were resuspended in the cell binding solution (300 μL) and analyzed by FCM (FACS Calibur and Celesta, BD, Biosciences) and FlowJo_V10 software.

Liposomes averagely sized 95 ± 25 nm were loaded with fluorescent dyes, rare earths or nutritional agents. The biodistribution of nanoparticles (NPs) and subsequent release rates of the compounds at the biological site were demonstrated on Cherry Tomato (Solanum Lycopersicum var. cerasiforme) by both qualitative and quantitative approaches. Samples were taken from different parts of the plant at predetermined time intervals post application. Visualization of biodistribution and capacity to penetrate the cell wall was performed using Confocal Laser Scanning Microscopy (Olympus LSM 510 Meta, Tokyo, Japan). Quantification of uptake extent was determined by Inductively-Coupled-Plasma Optical-Emission-Spectrometry (ICP-OES, Agilent 5100VDV, Santa-Clara, USA).

TEM and confocal laser scanning microscopy (CLSM) were employed to reveal the distributions of nanoparticles in cells. Cell seeding procedure was the same as above. Cells were co-cultured with HMSNs and HMSNs–S–S–CPA–CytC–LA (0.136 mg/mL) for 12 h. Cells culturing in tissue culture polystyrene (TCPS) without adding any nanoparticles was used as control. TEM samples were prepared as previous reports [24 (link), 25 (link)]. Cells were collected and fixed with glutaraldehyde (2% w/v) and paraformaldehyde (2% w/v) at 4°C for 2 h. Then, the samples were immersed into osmic acid (2%) for 15 min and stained with a uranyl acetate solution for another 15 min. The samples were dehydrated in a graded series of ethanol, followed by incubated with a mixture solution of dehydrated ethanol and Spurr's medium (1:1, v/v) for another 1 h. The samples were dried, cut into ultrathin sections and stained with uranyl acetate on the grid for 5 min. The images were recorded by a TEM microscope. For CLSM imaging, HMSNs@FITC and HMSNs–S–S–CPA–CytC–LA@FITC (0.136 mg/mL) were co-cultured with HepG2 cells for 6, 12 and 24 h, respectively. Next, cells were fixed with para-formaldehyde (4% w/v) at 4°C for 40 min. After that, the treated cells were stained by DAPI (20 µg/mL) and then washed 3 times with PBS. Fluorescence images of cells were acquired by CLSM (LSM 510 META; Olympus, Japan).

Fluorescent images were obtained using a fluorescent microscope (BX-51, Olympus) with the 10× objective. Confocal microscope images of various brain regions were generated using either the 63× objective and the Zeiss LSM 510 Meta or the 2× objective on the Olympus BX61. Image processing was done using the LSM 510 imaging software (Zeiss) or Slidebook 4.2. Merged images were created using Photoshop CS4 with the photomerge reposition feature.