Lsm510 confocor2

HeLa cells and EMT-6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), respectively. The media was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin in 5% CO₂ at 37°C in a humidified incubator. Before each experiment, the cells were precultured until confluence was reached. For the examination of cell imaging potency for PEG-TPA, EMT-6 cells were cultured for 4 h and then imaged by a commercial laser scanning microscope (LSM 510/ConfoCor 2, Zeiss, Jena, Germany). The fluorescent channel was recorded at the excitation wavelength of 405 nm. Secondly, for the imaging by Tat-TPA against EMT-6 cells, EMT-6 cells were incubated with Tat-TPA and TPA for 10 min, respectively, and then imaged by CLSM. Finally, for the imaging potency of three kinds of three small molecules, TPA, alkyne-TPA, and the conjugation product of alkyne-TPA with n-butylamine, these three molecules were treated with HeLa cells for 2 min in PBS, then washed with cold PBS, and imaged immediately by the CLSM system, respectively.

Subcellular localization of PlHSP70 was determined using confocal laser microscopy in O. sativa protoplasts. PlHSP70 product was obtained by constructing gene fusions of p35S::PlHSP70-GFP. The open reading frame (ORF) of PlHSP70 was amplified with primers that included BsaI/Esp3I restriction sites (forward 5’-CAGTCGTCTCACAACATGGCAGGCAAAGGAGAAGG-3′, reverse 5’-CAGTCGTCTCATACAGTCGACCTCTTCAATCTTGG-3′), which was digested and the product ligated into the expression vectors with T4 DNA ligase (TaKaRa) to generate a set of pBWA(V)HS-PlHSP70-GFP fusion, subsequently sequenced for verification. The p35S::PlHSP70-GFP constructs and the empty pBWA(V)HS-GFP vector were transformed into O. sativa protoplasts using a modified procedure that was described previously [22 (link)]. Gently mix 200 μL protoplast suspension, 10 μL plasmid DNA and 10 μL marker plasmid DNA together with 220 μL PEG solution, standing for 30 min at room temperature. After concentration at 100×g for 5 min, after which the precipitate was suspended in W5 medium. The transformed protoplasts were then incubated at 28 °C for 24 h in W5 medium in the dark. The transient expression of GFP and mkate were monitored by confocal laser microscopy combination system (LSM510/ConfoCor2, Zeiss, Germany).

HeLa cells were seeded into 35-mm Petri dishes and incubated for 24 h. The cell culture medium was removed and the DMEM containing the functional SWCNTs was added into each well, followed by incubation at 37 °C for 2 h. After incubation, HeLa cells were washed by changing the fresh DMEM and then characterized by confocal microscopy with a commercial laser scanning microscope (LSM 510/ConfoCor 2) combination system (Zeiss, Jena, Germany) equipped with a Plan-Neofluar 40×/1.3 NA Oil DIC objective. The excitation wavelength and detection filter settings for each of the fluorescence indicators were as follows. FITC was excited at 488 nm with an Ar-ion laser, and the fluorescence emission was recorded through a 500–530 nm band-pass filter. PPa was excited at 633 nm with a He-Ne laser, and the fluorescence emission was recorded through a 650 nm long-pass filter.

Cells were cultured on a confocal dish. After treatment with 3, 13, and 45 nm AuNPs for 8 h, the cells were washed with PBS and incubated with Mitotracker Deep Red 633 (0.1 μM, final concentration) for 30 min at room temperature in dark. The cells were then washed with PBS and visualized under confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany). Mitotracker Deep Red 633 was excited at 633 nm, and the emitted light was recorded through a 650-nm long-pass filter.

A549 and PC-9 cells were treated with indicated concentrations of DHA and 2DG for 48 h. Imaging of Living Cells was performed on a confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany) with a 40×oil immersion plan apochromat objective lens. Images were recorded using a digital camera with 1280×1280 pixels resolution.

Rho 123 was used to analyze ΔΨm by FCM as described previously [26 (link), 46 (link)]. Cells were stained with Rho 123 (6 μM, final concentration) for 30 mins at 37 °C in dark, and then washed with PBS, suspended by trypsinization, harvested by centrifugation (4 °C, 3000 RPM, 5 min), resuspended in 1 ml PBS and subsequently assayed by FCM with 488 nm excitation and 515 nm emission. Values of cellular fluorescence were obtained using FCS Express Version 3.
The ΔΨm in single living cells was also monitored in real-time by time-lapse confocal imaging as described previously [46 (link)]. Briefly, the fluorescence images of cells stained with 6 μM Rho 123 were monitored in real-time using a confocal microscope (LSM510/ConfoCor2, Zeiss, Jena, Germany) equipped with a device sustained culture condition (37 °C, 5 % CO₂) .

We cloned the 1–516 bp CDS of PavDAM1, full length CDS of PavDAM5 and PavSOC1 into the vector pXY104 and pXY106 to construct PavDAM1- pXY106, PavDAM5-pXY106 and PavSOC1-pXY104 for BiFC assay. Constructed vectors were transformed into Agrobacterium tumefaciens strain GV3101 and subsequently cultured to an OD600 of approximately 0.8–1.0. The mixed suspension liquid with pairs were co-transformed into five-week-old leaves of Nicotiana benthamiana after 2 to 5 h. Yellow fluorescent protein (YFP) signals were detected after 48–72 h by a laser scanning confocal microscope (Zeiss LSM510/ConfoCor2). Both pXY104 and pXY106 empty vectors were used as the controls.