Lsm510 axiovert 200m confocal microscope

NIH3T3^3c cells were plated in 4 well poly-L-lysine coated glass bottom dishes (D141410, Matsunami Glass Ind.) and reverse transfection was performed using Lipofectamine^® RNAiMAX (Invitrogen) with FoxO3 siRNA, FoxO6 siRNA or siRNA control. After 24 h, transfection medium was replaced with regular culture medium and placed in a temperature and humidity-controlled chamber (37 °C, 5% CO₂) of a live cell imaging Zeiss LSM510/Axiovert 200M confocal microscope. Images were then recorded every 30 min for 72 h or more using a Coolsnap HQ/Andor Neo sCMOS camera. Live cell imaging was conducted and analysed as described previously [32 (link),60 (link)]. Briefly, time series for each of the fluorescent markers (Rev-ERbα-VNP, hCDT1-mKOrange, hGeminin-CFP) were generated and used to assess lengths of the circadian cycle, total cell cycle, G1 phase and combined S, G2 and M phases (S/G2/M). The G1 phase was defined as the interval between the peaks of hGeminin-CFP and hCDT1-mKOrange expression whereas the S/G2/M phase was defined as the interval between the peaks of hCDT1-mKOrange and hGeminin-CFP expression.

QM5 cells grown to approximately 50% confluence on coverslips were transfected with FLAG- or Myc-tagged versions of ARV or MdRV p10 constructs, as described above. At 24 h post-transfection, cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h, and then incubated with 1:200 dilutions of mouse anti-FLAG and rabbit anti-Myc monoclonal antibodies overnight at 4 °C. Cells were thoroughly washed with PBS and then incubated with 1:1000 dilutions of Alexa 488-conjugated goat anti-mouse antibody and Alexa 555-conjugated goat anti-rabbit antibody for 1 h at room temperature. Coverslips were then mounted using prolong gold anti-fade reagent (Invitrogen) and imaged using a Zeiss LSM510 Axiovert 200M confocal microscope.

Confocal fluorescent images were taken using an inverted Zeiss LSM510 Axiovert 200M confocal microscope with a 63× oil-immersion objective. Sequential excitation wavelengths at 488 nm and 543 nm were provided by argon and helium-neon gas lasers, respectively. Emission filters BP500-550 and LP560 were used for collecting green and red images in channels one and two, respectively. After sequential excitation, green and red fluorescent images of the same cell were saved with ZEN2012 software. In some experiments, autofluorescence (green) detectable within the excitation wavelength of 480 nm is an indicator of lipofuscin accumulation. Images were analyzed by Zeiss software. Intensity of fluorescent was analyzed using ImageJ, The image size was set at 1,024 × 1,024 pixels.