China
Africa
Explore chapters and articles related to this topic
Scanning Angle Interference Microscopy (SAIM)
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Cristina Bertocchi, Timothy J. Rudge, Andrea Ravasio
SAIM is feasible for fixed samples as well as live-cell imaging. To avoid loss of accuracy we do recommend special attention to the choice of fluorophores. Although SAIM does not require special fluorophores, the fluorophores should have high photostability to minimize photobleaching during the imaging scanning sequence and should be bright with high quantum yield to provide a good signal-to-noise ratio. Among the fluorescent proteins and synthetic dyes compatible with SAIM, genetically encoded fluorescent proteins have the principal advantage of being small and suitable for live-cell imaging and are capable of achieving maximal labeling specificity, removing any possible problems associated with nonspecific labeling. Furthermore, they do not require fixation or permeabilization procedures that could affect cellular nanostructure. Some fluorescent proteins successfully used in SAIM include green fluorescent proteins such as EGFP and mEmerald, red fluorescent protein mCherry,33 and photoconverted tdEOS25 that has excellent brightness and photostability. In addition, chromobodies (generated by the fusion of a fluorescent protein to a nanobody34), a novel species of extremely small antibodies that are endogenously synthesized within cultured cells, could possibly be used to prepare samples for SAIM.
Preclinical evaluation of multimodality probes
Published in Yi-Hwa Liu, Albert J. Sinusas, Hybrid Imaging in Cardiovascular Medicine, 2017
Fluorescent proteins, e.g., green fluorescent protein (GFP) and red fluorescent protein (RFP), are among the earliest and well-established optical reporter probes that have been widely used primarily for in vitro gene expression identification and postmortem histological verification. GFP derived from jellyfish Aequorea victoria has been used to identify the presence of transplanted bone marrow- and adipose tissue-derived MSCs in the infarcted mouse myocardium (van der Bogt et al. 2009). However, fluorescent reporter proteins have inherent limitations, i.e., significantly high autofluorescence background and scattered photon attenuation. Although fluorescence techniques, such as fluorescence-mediated molecular tomography, which permits tomographic reconstruction, improved the detection depth up to 1 mm (Graves et al. 2003), it is unlikely that such limited penetration depth will be enough to allow in vivo cardiac imaging in large animals or man.
Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Recent mutagenesis experiments resulted in relatively stable true far-red fluorescent proteins (mPlum 590/649 and AQ143 590/655 from Actinia equina) that can be expressed in live cells (30,31). These proteins have more favorable light scattering/absorption profiles for in vivo imaging. Recently engineered monomeric Keima red fluorescent protein (32) with a large Stokes shift is useful in designing fusion reporters based on cross-correlation fluorescence spectroscopy, which is extremely sensitive to protein-protein interactions. However, the excitation band of 450 nm is less useful for in vivo applications. The disadvantage of these far-red proteins, including engineered monomeric fluorescent proteins is low quantum yield (brightness), which is 10% or even lower than that of EGFP. One member of phycocyanin protein family (allophycocyanin, a 100-kDa protein with six bilin fluorochromes per protein molecule) has maximum emission of 660 nm and extremely high photostability. However, in vivo use of phycocyanins is limited by their cost, potential high immunogenicity, and high molecular mass. As in the case of any other protein-based exogenous probe, the investigators should be aware of immune response to fluorescent proteins that can significantly alter the progression, therapeutic response, and regrowth of experimental tumors expressing fluorescent proteins (33).
Versatile cationic liposomes for RIP3 overexpression in colon cancer therapy and RIP3 downregulation in acute pancreatitis therapy
Published in Journal of Drug Targeting, 2020
Lijing Zhang, Simeng Liu, Huimin Liu, Chengli Yang, Ailing Jiang, Heng Wei, Dan Sun, Zheng Cai, Yu Zheng
Cells were seeded in 6-well cell culture plates at a concentration of 1 × 105 cells per well the day before transfection. After incubation for 24 h, the culture medium was replaced with 800 µL of serum-free DMEM in each well. The liposome/plasmid complexes in a final volume of 200 µL containing 1 µg of RFP-pDNA were subsequently added to the wells. After 5 h of incubation, the cell culture medium was again replaced with complete cell culture medium, and the cells were incubated for an additional 43 h. Expression of red fluorescent protein (RFP) was observed under an Olympus IX-70-fluorescence microscope (Tokyo, Japan) with a dichroic filter set (ex = 555 nm, em = 584 nm), and the transfection efficiency was compared. Three independent experiments were performed.
CRISPR/Cas9-based liver-derived reporter cells for screening of mPGES-1 inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Zhanfei Chen, Xiaoling Cai, Man Li, LinLin Yan, Luxi Wu, Xiaoqian Wang, Nanhong Tang
Human mPGES-1 is encoded by the PTGES (prostaglandin E synthase) gene and can be induced by the proinflammatory cytokine IL-1β. After treatment with IL-1β (2.5 ng/mL), the expression level of mPGES-1 mRNA increased (Figure 3(A)), and FCM results showed that the PE intensity was enhanced (Figure 3(B)). Two pairs of siRNAs (siRNA352 and siRNA271) were designed for the PTGES gene. siRNA was transfected into BEL-7404 WT cells, and protein was extracted 48 h after transfection. Western blot indicated that siRNA352 and siRNA271 had the PTGES knockdown effect (Figure 3(C)), but the effect of siRNA352 (knockdown by 74%) was more effective than that of siRNA271. Two pairs of siRNAs were transiently transfected into reporter cells. After 72 h, the expression of red fluorescent protein was observed via fluorescence microscopy. The red fluorescence was found to be considerably attenuated in the reporter cells transfected with siRNA compared with normal reporter cells (Figure 3(D)). The enhancement of fluorescence intensity by IL-1β and the inhibitory effect of siRNA also fully confirmed the accurate insertion of the fluorescent tag.
A novel strategy for in vivo angiogenesis and osteogenesis: magnetic micro-movement in a bone scaffold
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Cong Luo, Xiaolan Yang, Ming Li, Hua Huang, Quan Kang, Xiaobo Zhang, Hui Hui, Xin Zhang, Chaode Cen, Yujia Luo, Lina Xie, Changxuan Wang, Tongchuan He, Dianming Jiang, Tingyu Li, Hong An
HUVEC-1 and human osteogenic sarcoma cells (MG-63) were seeded onto 12-well cell culture plates at 1 × 105 cells per well. After 24 h of culturing, gene transfection was conducted, with a negative control for both types of cells. The prepared SPFCN was added to the sample wells, with 20 μl per well. The naked plasmid was added to the negative control wells. Then, the cell culture plate was placed in a CO2 incubator for culture, and the medium was replaced with 1 ml fresh medium every 48 h [18,23,24]. The cells were routinely observed for the generation of red fluorescent protein using inverted fluorescence microscopy. Four days after the transfection, the transfection efficiency of the plasmid was determined by flow cytometry. Some cells were fixed with 4% glutaraldehyde and observed using TEM.