Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of l...Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.展开更多
目的研究人骨随间充质干细胞(hum an m esenchym al stem cells,hMSCs)的细胞间缝隙连接通讯(gap junc-tional intercellu lar commun ication,G JIC)功能。方法透射电镜观察细胞缝隙连接结构,应用荧光光漂白恢复(fluorescencered istri...目的研究人骨随间充质干细胞(hum an m esenchym al stem cells,hMSCs)的细胞间缝隙连接通讯(gap junc-tional intercellu lar commun ication,G JIC)功能。方法透射电镜观察细胞缝隙连接结构,应用荧光光漂白恢复(fluorescencered istribution after photob leach ing,FRAP)技术,5,6-CFDA荧光负载hMSCs,激光淬灭细胞内荧光,通过激光共聚焦显微镜测定体外培养的hMSCs的G JIC功能。结果细胞内荧光被淬灭后可通过缝隙连接通道恢复,hMSCs平均荧光漂白恢复率为(35.26±0.76)%。结论缝隙连接是间充质干细胞间的重要通讯方式。展开更多
激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 n...激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。展开更多
AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7...AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay. RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）. CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.展开更多
Objective: To demonstrate the functional changes of gap junctional mediation of intercellular communication in detrusor instability (DI) and its mechanisms. Methods: The function of gap junctional intercellular commun...Objective: To demonstrate the functional changes of gap junctional mediation of intercellular communication in detrusor instability (DI) and its mechanisms. Methods: The function of gap junctional intercellular communication in the cultured bladder detrusor cells was detected by fluorescence redistribution after photobleaching. Results: At the fourth minute after bleaching, the mean fluorescences recovery rates of DI group bladder detrusor cells were (35 791±0 836)%, that of control group (8 645±0 673)%. The mean fluorescence recovery rates of DI group were significantly higher than those of control group ( P <0 01). Conclusion: It shows that the increase of intercellular excitatory communication is one of the important reasons of pathogenesis of DI.展开更多
基金This work was supported by grants from the National Research Foundation(NRF)(NRF2019R1A2C2088973)funded by the Ministry of Educationthe Korea Evaluation Institute of Industrial Technology(KEIT)(20011377)funded by the Ministry of Trade,Industry&Energy,Republic of Korea.
文摘Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.
文摘目的研究人骨随间充质干细胞(hum an m esenchym al stem cells,hMSCs)的细胞间缝隙连接通讯(gap junc-tional intercellu lar commun ication,G JIC)功能。方法透射电镜观察细胞缝隙连接结构,应用荧光光漂白恢复(fluorescencered istribution after photob leach ing,FRAP)技术,5,6-CFDA荧光负载hMSCs,激光淬灭细胞内荧光,通过激光共聚焦显微镜测定体外培养的hMSCs的G JIC功能。结果细胞内荧光被淬灭后可通过缝隙连接通道恢复,hMSCs平均荧光漂白恢复率为(35.26±0.76)%。结论缝隙连接是间充质干细胞间的重要通讯方式。
文摘激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。
基金Supported by Natural Science Fund of Zhejiang Province,No.302023
文摘AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay. RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）. CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.
文摘Objective: To demonstrate the functional changes of gap junctional mediation of intercellular communication in detrusor instability (DI) and its mechanisms. Methods: The function of gap junctional intercellular communication in the cultured bladder detrusor cells was detected by fluorescence redistribution after photobleaching. Results: At the fourth minute after bleaching, the mean fluorescences recovery rates of DI group bladder detrusor cells were (35 791±0 836)%, that of control group (8 645±0 673)%. The mean fluorescence recovery rates of DI group were significantly higher than those of control group ( P <0 01). Conclusion: It shows that the increase of intercellular excitatory communication is one of the important reasons of pathogenesis of DI.
