REF52 WT, REF52E2VF1 (clone 8), REF52 E2VF1–3′UTR (clone D) cells were imaged under brightfield or DIC illumination and YFP illumination (20×) to confirm expression and localization of the fusion protein. For time-lapse microscopy, quiescent cells growing in 35 mm p35 Mattek optic plates were released into the cell cycle and placed into the Olympus VivaView FL incubator microscope. Images were taken every 30 min for 36 h using a 20×0.75 DIC Olympus UPlanSAPO 0.65 mm WD objective lens under DIC illumination and YFP 25% illumination (150 msec). Quiescent HMEE2Fact Rb+, E2VF1–3′UTR (clone R15) and HMEE2Fact Rb−, E2VF1–3′UTR (clone H4) cells were released into the cell cycle and placed into Olympus VivaView FL incubator microscope. Images were taken every 30 min for 40 h using a 20×0.75 DIC Olympus UPlanSAPO 0.65 mm WD objective lens under DIC illumination (30 msec), RFP 25% illumination (300 msec), YFP 25% illumination (1,000 msec) (binning 2). For time course fluorescence quantification, images were analyzed in ImageJ and values were determined as previously described [8 (link)].
Vivaview fl incubator microscope
Manufactured by Olympus
Sourced in Canada
The VivaView FL incubator microscope is a live-cell imaging system designed for long-term cell culture observation. It provides a controlled environment for cells, maintaining optimal temperature, humidity, and gas composition. The microscope is equipped with fluorescence imaging capabilities, allowing users to visualize and track cellular processes over extended periods.
Automatically generated - may contain errors
Lab products found in correlation
Cell tak adhesive, by BD (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Uplansapo 0.65 mm wd objective lens, by Olympus (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Chir99021, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Glass bottom culture dish, by MatTek (1 mentions)
10 protocols using vivaview fl incubator microscope
1
Time-lapse Imaging of Cell Cycle Progression
2
Visualizing Virus Infection Dynamics
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Cell-Tak adhesive (BD Biosciences) coated glass-bottom dishes were prepared on the day of use by adsorption with sodium bicarbonate and NaOH. MT4 cells, infected at a sufficiently low MOI so that each infected cell contained a single provirus were dispensed into these plates after washing, centrifuged at 350g for 8 minutes and immediately transferred to the microscope incubator. Time-lapse microscopy was performed using a VivaView FL incubator microscope (Olympus). Images of infected MT-4 cells were captured every 2–5 minutes using GFP, RFP, and DIC filter sets. Preparation of movies and quantitation of microscope data was done using Metamorph software (Molecular Devices). For quantitation, regions were drawn closely around immobilized cells and the maximum fluorescence intensity within each region was logged in both the GFP and mCherry channels. Fluorescence in unoccupied regions proximal to each cell was also quantified to provide background subtraction values for each cell. The background-corrected fluorescence intensities were analyzed using a custom MATLAB script that fit each set of fluorescent intensity data to a 5-parameter sigmoid logistic function. The function was used to identify time points where the signal was 7 intensity units above the baseline level. (Images had a bit-depth of 12, i.e., an intensity range of 0–4095).
3
Visualization of Cav-1 Mutant Dynamics
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MDA-MB-231 or MCF-7 cells transfected with CFP-tagged Cav-1 WT, Y14D, Y14F mutants were plated into glass-bottom dishes at a density of 5000 cells per well. After 24 h, time-lapse images were acquired using an Olympus VivaView FL Incubator Microscope every 6 min for 4 h. Image sequences were analyzed using ImageJ software with a manual tracking plugin.
4
NPC Proliferation Monitoring Using Microscopy
5
Neutrophil Transendothelial Migration Assay
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After 69–72 hours of siRNA treatment, ECs were trypsinized and replated onto FN-coated 35mm glass-bottom dishes at confluent density (450k cells per dish). At 6–7 hours post replating, ECs were treated with 10ng/mL TNFα for 18–20 hours and transferred to a Viva View FL Incubator Microscope (Olympus) for phase contrast imaging. Freshly isolated neutrophils were activated by 10-minute-incubation at 37°C. Neutrophils (2.2 x 105 cells/dish) were added to each 35mm dish, and neutrophil TEM was imaged every 15–30 seconds for 30 minutes.
6
Visualizing Mitosis and Cytokinesis in A549 Cells
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H2B-mCherry was a gift from Robert Benezra (Addgene plasmid # 20972)57 (link). A549 cells stably expressing H2B-mCherry were selected using G418 (800 μg/ml) for 14 days. The cells were then silenced by COASY or control siRNAs. The phenotypes of extended mitosis and cytokinesis failure were assessed in living cells using Olympus VivaView FL incubator microscope. The images of DIC and mCherry were taken every 6 min.
7
Quantifying NPC Proliferation Dynamics
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NPC proliferation was assessed by cell counting. Briefly, a pre-determined number of NPCs was plated onto poly-L-ornithine/laminin-coated plates (day 0). After 4 h, the plates were transferred to a Viva View FL Incubator Microscope (Olympus), and allowed to acclimatize for 30 min (T0). Next, the cellular proliferation was monitored and contrast-phase images were taken after 48 h (T48; day 2). The images were processed, and the number of cells at T0 and T48 was determined using the Cell Counter plugin on the Fiji platform. The difference between days 2 and 0 was used to estimate the NPC proliferation rate.
8
Astrocyte Morphology Dynamics
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Astrocyte cultures were plated on 35 mm glass bottom dishes from MatTek Corporation (Ashland, MA, USA) and grown for 2 days. 1 μg/ml of LPS was added into the media for 48 hours followed by 10 μM of GluA2-G-Gpep. Differential interference contrast (DIC) images were captured using the Vivaview FL incubator microscope (Olympus, Toronto, Canada). The recording parameters for image capturing were set at 10-minute intervals for 60 hours for analysis of astrocyte morphology.
9
Sox2-Expressing Cochlear Explant Culture
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Cochlear explant cultures from E13.5 Sox2EGFP/+ reporter mice were established and imaged as previously described using an Olympus VivaView FL Incubator Microscope (Mulvaney and Dabdoub, 2014 (link)). The Wnt pathway was activated with CHIR99021 (1.5 μM; EMD Millipore) for 5 DIV. Cochlear explants of littermates were cultured with DMSO as a control.
10
Visualizing Megakaryocyte Maturation and Platelet Release
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BM explants were obtained by flushing femurs, cutting into 1 mm pieces, and staining with anti–GPIX-AF488 antibody (10 μg/mL) for 30 minutes. Explants were then placed in a glass-bottom culture dish (MatTek) precoated overnight with fibronectin (20 μg/mL; MilliporeSigma) with Tyrode’s buffer (137 mM NaCl; 2 mM KCl; 0.3 mM NaH2PO4; 5.5 mM glucose; 5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; 12 mM NaHCO3; 2 mM CaCl2, pH 7.4) and maintained at 37°C in a VivaView FL Incubator Microscope (Olympus). Images were acquired every 5 minutes in differential interference contrast and GFP channels for 18 hours to visualize MKs released at the periphery of the explants. Videos were analyzed using ImageJ (NIH).
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