Recombinant protein assay images were acquired on an inverted fluorescent Nikon Eclipse Ti2-E microscope equipped with an Andor Zyla 5.5 sCMOS camera and a Lumencor SOLA-SE II light engine. The objectives used were CFI Nikon PlanApo Lambda ×20 with 0.75 NA and CFI Nikon Plan Apo Lambda ×60 oil lenses with 1.40 NA and a ×1.5 adapter. Acquisition settings were kept consistent across all conditions within a given experiment. For high magnification, mixed culture assay images were taken on a Zeiss Cell Observer SD confocal microscope with a Yokagawa CSU-X1 spinning disk, Plan-Apochromat ×63/1.4 M27 objective paired with a ×1.2 adapter to a Photometrics Evolve 512 EMCCD camera that was used for image acquisition. The laser lines used were 405 nm, 488 nm, 561 nm, and 639nm.
Eclipse ti2 e microscope
Manufactured by Oxford Instruments
The Eclipse Ti2-E microscope is a high-performance inverted fluorescence microscope designed for advanced live-cell imaging and analysis. It features a stable, vibration-free optical system, high-resolution objectives, and a wide range of accessories to support various experimental requirements. The Eclipse Ti2-E allows researchers to capture high-quality images and perform precise measurements of cellular processes and structures.
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Lab products found in correlation
Sola se 2 light engine, by Lumencor (1 mentions)
Plan apo lambda 60, by Nikon (1 mentions)
5.5 scmos camera, by Oxford Instruments (1 mentions)
Nis elements software, by Nikon (1 mentions)
Histofix, by Carl Roth (1 mentions)
C3100mp, by Thermo Fisher Scientific (1 mentions)
4.2 scmos camera, by Oxford Instruments (1 mentions)
Matlab, by MathWorks (1 mentions)
5 protocols using eclipse ti2 e microscope
1
Fluorescent Microscopy for Protein Assays
2
cAMP Imaging in Transfected Cells
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cAMPr imaging was performed 24-48 hours after transfection at room temperature on an inverted fluorescent Nikon Eclipse Ti2-E microscope equipped with an Andor Zyla 5.5 sCMOS camera. Briefly, movies were the acquired with 100 ms exposures at 0.25-0.5Hz with 20x objective. Cells were continuously perfused with the same extracellular solution same as used in HEK 293 calcium imaging experiments. BDNF or DAMGO was applied to cells 2 min prior to Forskolin co-application. Fluorescence intensity was analyzed in similarly, as previously described for HEK 293 calcium imaging. Area under the curve (AUC) of the normalized traces was used to quantify the cAMP response. Analysis was performed using ImageJ and NIS-Elements Advance Research 5.2.6 software. All analyses were manually performed on Microsoft Excel, with statistical analysis performed on GraphPad Prism
3
Epifluorescence Microscopy Protocol for Cell Nuclei
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Roots were fixed in 4% Histofix (phosphate‐buffered formaldehyde solution; Carl Roth) for one hour. Samples were dehydrated in an ascending ethanol series (50% EtOH, 2 × 70% EtOH, 100% EtOH) before being incubated in Hoechst 33342 for 10 min, mounted in Vectashield (H‐1000, Vector Laboratories), and analyzed using epifluorescence microscopy. Alternatively, propidium iodide was used for DNA staining on Histofix‐preserved samples by mounting them directly with ROTI®Mount FluorCare PI (Carl Roth). Samples were analyzed using a Nikon Eclipse Ti2‐E microscope equipped with an Andor Zyla 5.5sCMOS monochrome camera and Nikon CFI Plan‐Fluor 40×/0.75 NA and 60×/0.85 NA objectives using the excitation wavelengths 365 nm (for Hoechst 33342) and 535 nm (for propidium iodide). The NIS Elements software (Nikon) was used for imaging analysis (overlaying images from the DIC channel with images from the fluorescent channels for Hoechst or PI). The image plates presented in this paper were assembled using Inkscape 0.92.4.
All measurements are given in the form (minimum) mean ± standard deviation (maximum).
All measurements are given in the form (minimum) mean ± standard deviation (maximum).
4
Organoid-based Assessment of CFTR Rescue
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After 6–7 days of culture in Matrigel or various Trpzip gels, organoids less than 100 µm in size were selected and seeded in 4 μL Matrigel droplets in a 96-well plate. For viability assessment, organoids were incubated with Calcein AM (5 µM; Thermo Fisher Scientific C3100MP) for 30 min prior to addition of forskolin (5 μM). For cystic fibrosis transmembrane conductance regulator (CFTR) targeted correction, Trikafta (VX-770 + VX-661 + VX-445) was added together with forskolin. Organoid swelling was monitored using time-lapse brightfield imaging (acquired at 10 min intervals for 90 min) using a Nikon Eclipse Ti2-E microscope coupled with a Andor Zyla 4.2 sCMOS camera. A custom-built script was used to quantify organoid swelling. Total organoid surface area post-forskolin exposure was calculated and normalized against initial organoid surface area to determine the relative amount of swelling. The area under the curve (calculated increase in organoid surface area from t = 0 to t = 90; baseline=100%) was then calculated using GraphPad Prism.
5
Quantifying Cilia Beating in hNECs
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Cilia beating in differentiated hNECs was imaged and analysed as previously described (Allan et al., 2021 (link)). Imaging was performed using a Nikon Eclipse Ti2-E microscope with an Andor Zyla 4.2 sCMOS camera and a CFI S Plan Fluor ELWD 20×/0.45 objective. Time-lapse images were acquired from six fields of view from each of n = 2–3 differentiated hNEC cultures per participant. Cilia beat frequency (CBF) was analysed using a custom-built script in Matlab (MathWorks, Natick, MA) that identified the dominant frequency (highest peak).
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