For FRAP experiments, RPE1‐Tet3G cells constitutively expressing SSTR3‐GFP and γ‐Tubulin‐mRuby2 were cultured in HEPES‐buffered DMEM/F12 supplemented with 10% FBS, 1% l ‐glutamine and 1% penicillin–streptomycin without phenol red (ThermoFischer) in glass‐bottom CELLview culture dishes (Greiner Bio‐One) for 24 h and serum‐starved for 32 h. Dishes were mounted in a pre‐heated microscope chamber and live cell imaging was performed over a period of 10 min at 37°C with 5% CO2. Images were acquired by the Nikon TIRF/FRAP microscope system mounted with the Hamamatsu Orca AG camera and a Nikon Plan Apo λ 100× NA 1.45 oil immersion objective every 30 s. Image acquisition was processed in Nikon NIS‐Elements Imaging Software. Acquired images were analysed using Fiji. Cilia length (µm) and the integrated density of cilia area (IntDenCilia) were measured each time point. The IntDenCilia was normalised by the cilia length and subtracted IntDenCilia after bleach. Percentage of FRAP = normalised IntDenCilia at each time point/normalised IntDenCilia before bleach.
Cellview culture dishes
Manufactured by Greiner
Sourced in France
CELLview culture dishes are sterile, transparent cell culture vessels with a grid pattern. They are designed for the cultivation and microscopic observation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Nis elements imaging software, by Nikon (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Fitc labelled collagen, by Merck Group (1 mentions)
Lamp1, by BioLegend (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
4 protocols using cellview culture dishes
1
Cilia FRAP Dynamics in RPE1 Cells
2
Live-cell imaging of LPS-stimulated macrophage phagocytosis
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For live-cell imaging, cells were seeded (100,000 cells per quadrant) on Cellview culture dishes (Greiner bio-one, 627870) which were coated with 10 μg/ml FITC-labelled collagen (Sigma, C4361) in PBS for 1 h at room temperature in the dark. Cells were stimulated with LPS (100 ng/ml) for 24 h.
After collagen uptake in a 24 well plate (coated as described above) macrophages were seeded on glass coverslips in RPMI for 30 min to attach and fixed using 4% paraformaldehyde (PFA, 15 min at 4 °C) followed by four PBS washes. Cells were blocked and permeabilised for 30 min at 4 °C with CLSM buffer (PBS + 20 mM glycine + 3% BSA) and 0.1% saponin followed by an overnight staining with the primary antibody (Cathepsin B, Calbiochem IM27L and LAMP1, Biolegend 328601) 1:200 diluted in CLSM with saponin at 4 °C. The next day cells were washed twice with PBS +0.1% saponin and incubated for 30 min at room temperature in CLSM +0.1% saponin with secondary antibodies donkey-anti-mouse IgG (H&L) labelled with Alexa 647 and donkey-anti-rabbit with Alexa 568. After washing the cells with PBS with 0.1% saponin, the coverslips were mounted on glass slides in 67% glycerol containing Trolox (1 mM) and DAPI (0.33 μg/ml). Samples were imaged using an LSM800 Zeiss microscope with a 63 × oil immersion lens.
After collagen uptake in a 24 well plate (coated as described above) macrophages were seeded on glass coverslips in RPMI for 30 min to attach and fixed using 4% paraformaldehyde (PFA, 15 min at 4 °C) followed by four PBS washes. Cells were blocked and permeabilised for 30 min at 4 °C with CLSM buffer (PBS + 20 mM glycine + 3% BSA) and 0.1% saponin followed by an overnight staining with the primary antibody (Cathepsin B, Calbiochem IM27L and LAMP1, Biolegend 328601) 1:200 diluted in CLSM with saponin at 4 °C. The next day cells were washed twice with PBS +0.1% saponin and incubated for 30 min at room temperature in CLSM +0.1% saponin with secondary antibodies donkey-anti-mouse IgG (H&L) labelled with Alexa 647 and donkey-anti-rabbit with Alexa 568. After washing the cells with PBS with 0.1% saponin, the coverslips were mounted on glass slides in 67% glycerol containing Trolox (1 mM) and DAPI (0.33 μg/ml). Samples were imaged using an LSM800 Zeiss microscope with a 63 × oil immersion lens.
3
Live-cell imaging of ciliary dynamics
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For live‐cell imaging, cells were cultured in HEPES‐buffered DMEM/F12 supplemented with 10% FBS, 1% l ‐glutamine and 1% penicillin–streptomycin without phenol red (ThermoFischer) in glass‐bottom CELLview culture dishes (Greiner Bio‐One) for 24 h and serum‐starved for 32 h. Dishes were mounted in a pre‐heated microscope chamber and live cell imaging was performed over a period of 15 h at 37°C with 5% CO2. 3D‐Images (8 z‐stacks of 6 µm spacing) were acquired by the Nikon/Andor TuCam system mounted with the Andor Neo sCMOS camera and a Nikon Plan Apo VC 60× NA 1.4 oil immersion objective every 15 min. For chemical treatment, 200 nM Cytochalasin D or 100 nM nocodazole was added 30 min before imaging. For rescue experiments, 10 nM of doxycycline was added to induce EGFP‐SEPT2 protein expression when cells were initially seeded on CELLview culture dishes. Images were processed in Nikon NIS‐Elements Imaging Software using maximum intensity projections. The cilia length in each time‐point was manually measured using Fiji. For analysis of live cell imaging shown in Fig 7I and J , images were applied subtract background (rolling ball radius: 15 pixels) using Fiji to reduce background signal noise of Neongreen‐EFHC1.
4
Gastrin-Conjugated Magnetic Nanoparticle Internalization
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Cells were plated onto Cellview culture dishes (Greiner Bio-One, Courtaboeuf, France). After overnight growth, cells were incubated with Gastrin–MNPs ([Fe] = 16 μg/mL) in incubation medium (RPMI 1640 buffered with 10 mM HEPES buffer pH 7.4 containing 0.5% heat inactivated FBS and 100 units/mL penicillin-streptomycin) for 24 h. For lysosome staining, cells were incubated for 15 min in the presence of 75 nM LysoTracker Red DND-99 (excitation: 570 nm, Life technologies). Gastrin–MNP (excitation: 633 nm) and Lysotraker co-localization was analyzed using a LSM780 confocal microscope (Zeiss, Marly le Roi, France).
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