Tetrahymena thermophila cells expressing GFP-tagged proteins were collected by low-speed centrifugation and fixed with cold methanol for 30 min at −30 °C, and then further fixed with 4% formaldehyde for 30 min at room temperature (~25 °C). After washing three times with phosphate-buffered saline (PBS) for 10 min each, the fixed cells were counterstained with 0.05 μg/mL 4′,6‑diamidino‑2‑phenylindole (DAPI) and mounted between coverslips with 25% (v/v) glycerol in PBS. Fluorescence images were obtained using a fluorescence microscope IX-70 (Olympus, Tokyo, Japan) with an oil-immersion objective lens UApo 40×/1.35 oil or PlanApo N60×/1.40 oil (both from Olympus) equipped in the DeltaVision microscope system (GE Healthcare, Little Chalfont, UK). Twenty z-stack images at 0.5-μm intervals were acquired for each cell and deconvolved using softWoRx software (GE healthcare).
Deltavision microscope system
Manufactured by GE Healthcare
Sourced in United Kingdom, Japan, United States
The DeltaVision microscope system is a high-performance fluorescence imaging platform designed for advanced live-cell and fixed-sample imaging. The system features a robust optical design, precision control of sample environment, and powerful image acquisition and analysis software to enable detailed visualization and quantification of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Softworx software, by GE Healthcare (1 mentions)
Cellview cell culture dishes, by Greiner (1 mentions)
Uapo40, by Olympus (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Coolsnap hq2 ccd camera, by Teledyne (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Softworx package, by GE Healthcare (1 mentions)
Te300, by Nikon (1 mentions)
Glass bottom chamber slides, by Ibidi (1 mentions)
Apc labeled annexin a5, by BioLegend (1 mentions)
Can get signal immunostain solution a, by Toyobo (1 mentions)
P0067, by Merck Group (1 mentions)
Plapon60xosc na1, by Olympus (1 mentions)
Blocking one solution, by Nacalai Tesque (1 mentions)
Planapon60 osc, by Olympus (1 mentions)
Ix81 zdc, by Olympus (1 mentions)
Inub oni f2, by Tokai Hit (1 mentions)
Softworx, by Cytiva (1 mentions)
μ dish35 mm quad, by Ibidi (1 mentions)
14 protocols using deltavision microscope system
1
Fluorescence Imaging of GFP-Tagged Tetrahymena
2
FRET Analysis of Cellular Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FRET analysis was performed according to the previously published method with minor modification26 (link). Briefly, FRET signals were imaged with a DeltaVision microscope system (GE Healthcare) built on an Olympus IX-71 inverted microscope base equipped with a Photometric Coolsnap HQ2 CCD camera and a 60×/NA1.516 PlanApo oil immersion lens (Olympus). For live-cell imaging FRET sensors, cells were seeded on gelatin-coated CELLview Cell Culture Dishes (Greiner Bio-One) and maintained in an incubator at 37 °C with 5% CO2. For imaging, cells were observed with a Blue excitation filter (400-454 nm), two emission filters (blue-green, 463–487 nm for ECFP; yellow-green, 537–559 nm for Ypet), and a C-Y-m polychronic mirror. The FRET emission ratio (FRET/CFP) was calculated with SoftWoRx (Applied Precision Inc) by dividing the excitation at 436 nm and emission at 560 nm (FRET) by the excitation at 436 nm and emission at 470 nm (CFP). For statistical analyses, the obtained images were analyzed with ImageJ and MetaMorph software. The ΔFRET/CFP ratios were calculated by subtracting the FRET/CFP ratio at time 0 from the FRET/CFP ratio at the indicated times.
3
Visualizing Nucleoporin Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular localizations of GFP-tagged Nups were observed by performing fluorescence microscopy (IX-70; Olympus, Tokyo, Japan). Images were taken using the DeltaVision microscope system (GE Healthcare, Issaquah, WA) with oil-immersion objective lens UApo40 (NA=1.35) (Olympus). Line profiles of fluorescence intensity were obtained with a measurement tool included in the DeltaVision system. Background fluorescence was measured from the cytoplasm as an averaged value of 5×5 pixels and was subtracted from the peak values of fluorescence on the NE.
4
Imaging Neonatal Mouse Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neonatal mouse cardiomyocytes were isolated from the ventricles of 3-day-old mice homozygous for PLN R14del or wild-type C57Bl6/J littermates after decapitation. After trypsinization, cells were passed through a 40μm cell strainer and plated for 70 min to get rid of large debris and fibroblasts. Cells were plated on culture plates with microcontact printed flexible PDMS substrates at 50.000 cells per well and cultured at 37 °C under 5% CO2. Media for the first 24 h was MEM supplemented with 5% FCS and 1% penicillin–streptomycin, hereafter DMEM with 10% FCS and 1% penicillin–streptomycin was used. Media was additionally supplemented with 1% BrdU for the first 4 days. Cells where treated with either 15 μM PLN-ASO or PBS for 30 h. After an additional 24 h, cells were incubated with 2.5 μM Fluo-4 (F14201, Invitrogen) for 20 min at RT, followed three washes with warm PBS and placed in RPMI media with HEPES 1:1000. Cell were then paced at 3 Hz and live imaging was recorded at 74 fps using a DeltaVision Microscope System from GE Healthcare52 (link).
5
Fixed Cell Imaging with DeltaVision
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wide-field microscopy analysis of fixed and stained cells was performed on a Delta Vision microscope system (GE Healthcare) using a 60×UplanSApo objective. Images were acquired using Coolsnap HQ2 CCDcamera (Roper Scientific), SoftWorx software and were processed using ImageJ software.
6
Quantifying Chromosome Dynamics using Hoechst Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with 100 ng·mL−1 Hoechst33342 (B2261; Sigma‐Aldrich) for 15 min to stain chromosomes as previously described 25 . After replacing the culture medium with fresh medium not containing phenol red, time‐lapse observation was performed using an oil‐immersion objective lens (UApo40/NA1.35; Olympus, Tokyo, Japan) on a DeltaVision microscope system (GE Healthcare Life Sciences Japan, Tokyo, Japan) placed in a temperature‐controlled room (37 °C) as previously described 25 .
The fluorescence intensity around the beads was quantified using thefiji software suite (imagej ; National Institutes of Health, Bethesda, MD, USA). The fluorescence intensity of the region (18 pixels square) surrounding the beads was measured, and the background fluorescence intensity of a region with no beads (18 pixels square) in the same cell was subtracted. The fluorescence intensity was plotted as a function of time.
The fluorescence intensity around the beads was quantified using the
7
Live-Cell Fluorescence Imaging of Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A DeltaVision microscope system (GE Healthcare) was used to acquire live-cell fluorescence images as described previously (Li et al., 2015 (link)). Briefly, an agarose pad with 2% raffinose was prepared on a concave slide, and an aliquot of yeast cells was placed on the agarose. The slide was then sealed with LVP (Li et al., 2015 (link)), and images were acquired at each of the indicated time points at 30°C. Images were acquired with a 63× (NA = 1.40) objective lens on an inverted microscope (IX-71; Olympus) equipped with a CoolSNAP HQ2 CCD camera (Photometrics). Pixel size was set at 0.10700 µm. For images attained during time course experiments, 10 optical sections with a 0.5-µm thickness were acquired at each time point. To reduce phototoxicity, a neutral density filter was used to diminish the intensity of the excitation light to ∼50% or less of the equipment output. For GFP, the excitation spectrum was set at 470/40 nm, and emission spectrum at 525/50 nm; for mApple, excitation was at 572/35, and emission at 632/60 nm. Acquired images were deconvolved using the SoftWorx package (GE Healthcare). Projected images are used for display (Fig. 2, D and G ; and Figs. 6 I , S1, and S4).
8
Visualizing Cell Dynamics and Adhesion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DAPI-stained cells were observed using a fluorescence microscope (TE 300, Nikon, Japan) equipped with a regular UV filter set. Fluorescence images of live cells expressing GFP-dynamins and GFP-clathrin were acquired by a confocal microscope (LSM510, Zeiss, Germany) or a custom-made total internal reflection fluorescence (TIRF) microscope [31 (link)].
Traction force exerted by dividing cells was measured as previously described [22 (link)]. Briefly, cells were placed on an elastic silicone substratum with fluorescent red beads and observed using a DeltaVision microscope system (GE Healthcare, Little Chalfont, UK). To acquire the initial position-image of the beads, 10% sodium azide was added to kill the cells after the observation.
Interference reflection microscopy (IRM) was also simultaneously conducted using a DeltaVision microscope as previously described [22 (link)]. The cell-substratum adhesion area was measured using ImageJ software (http://rsbweb.nih.gov/ij/ ).
Traction force exerted by dividing cells was measured as previously described [22 (link)]. Briefly, cells were placed on an elastic silicone substratum with fluorescent red beads and observed using a DeltaVision microscope system (GE Healthcare, Little Chalfont, UK). To acquire the initial position-image of the beads, 10% sodium azide was added to kill the cells after the observation.
Interference reflection microscopy (IRM) was also simultaneously conducted using a DeltaVision microscope as previously described [22 (link)]. The cell-substratum adhesion area was measured using ImageJ software (
9
Fluorescence Microscopy of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence deconvolved images were taken using the DeltaVision Microscope system (GE Healthcare Life Sciences, Piscataway, NJ, USA). EVs were observed using glass bottom chamber slides (80807, Ibidi). Isolated EVs were incubated with 0.8 µg/mL APC-labeled annexin A5 (640919, BioLegend, San Diego, CA, USA) and 4.8 µg/mL FITC-GlaS for 30 min at room temperature and directly observed by fluorescence microscopy. The fluorescence filter set FITC/Cy5 and the 60x oil immersion objective, 1.42 NA, were used to acquire images and process z-stacks (Optical section space: 0.2 µm, Number of optical sections: 30) for deconvolution. Maximum intensity projection images of z-stack were created using ImageJ software (Version 1.53a, NIH).
10
Immunostaining of p62 in Murine Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine embryonic fibroblast cells grown in a glass‐bottom dish were fixed with 4% PBS for 15 min at room temperature. After washing with 0.1% Tween 20 in PBS (PBST), cells were permeabilized with 0.5% Triton X‐100 in PBS for 5 min at room temperature and washed with PBST three times. The cells were then blocked with Blocking One solution (03953‐95; Nacalai Tesque) for 30 min at 4 °C, washed once with PBST, and incubated with a rabbit polyclonal anti‐p62 antibody (P0067; Sigma‐Aldrich) in Can Get Signal Immunostain Solution A (NKB‐501; Toyobo, Osaka, Japan) for 2 h, followed by extensive washes and incubation with Cy3‐conjugated donkey anti‐rabbit IgG antibody (AP182C; Life Technologies, Carlsbad, CA, USA) for 1 h. After washing with PBST three times, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and subjected to FM. For observation, an oil‐immersion objective lens (PLAPON60xOSC/NA1.40; Olympus) on the DeltaVision microscope system (GE Healthcare Life Sciences) was used as previously described 25 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!