MDCK cells expressing mRFP-EB1 were seeded onto glass-bottomed imaging dishes (Mattek) pre-coated with either poly-D -lysine (Sigma, according to manufacturer's instructions) or E-cadherin:Fc (functionalized similarly as sidewalls, see above). Six hours after seeding cells in metaphase with the mitotic spindle oriented perpendicular to the basal surface were live-imaged by Total Internal Reflection Fluorescence (TIRF) on a Zeiss Observer inverted microscope (3I) through a 100x Plan Fluor objective (1.45 N.A) and a 561 nm solid-state laser (CristaLaser) in a temperature and CO2-controlled incubator. Images were taken at 1 s intervals for 60 s, and the dwell time of individual EB1 puncta within the TIRF plane (that were present for at least two consecutive frames) was measured using ImageJ software.
Glass bottomed imaging dishes
Manufactured by MatTek
Glass-bottomed imaging dishes are specialized laboratory equipment designed for live-cell imaging applications. These dishes feature a transparent glass bottom that allows for high-quality microscopic observation and analysis of cells or other biological samples. The glass bottom provides a clear, distortion-free surface for imaging, enabling researchers to study cellular processes, morphology, and behavior in a controlled environment.
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6 protocols using glass bottomed imaging dishes
1
Measuring EB1 dynamics in mitotic MDCK cells
2
Super-resolution Imaging of Integrin Nanoclusters
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mAb K20 was adsorbed on glass-bottomed imaging dishes (MatTek) for 30 min at RT and then diluted 105 times in PBS + 0.5% BSA to promote individual ab adsorption. Secondary ab (Alexa Flour 405/647 labeled) staining and STORM imaging settings used were identical as for ab-based cell labeling. Clusters in STORM images of mAb K20 on glass and mAb K20–labeled FAs in cells were identified by the DBSCAN algorithm (minimal number of points = 2; radius [ε] = 20 nm) integrated in Insight3. The mean numbers of localizations were compared with estimates of the number of integrin labels per nanocluster within FAs.
3
siRNA Knockdown of AP2M1, CAV-1, and FLOT-1
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siRNA sequences: Dharmacon ON-TARGET plus SMARTpool siRNAs were obtained from GE Healthcare targeting AP2M1 (L-008170-00-0005), CAV-1 (L-003467-00-0005) and FLOT-1 (L‑010636-00-0005). Non-targeting siRNA control, GFP (5'-GGCUACGUCCAGGAGCGCAdTdT-3') was synthesised by MWG.
SKBR3 cells (300,000) were seeded in 35 mm glass- bottomed imaging dishes (MatTek) and incubated for 24 hr in complete medium. The transfection mixture was prepared the following day by mixing: transfection reagent 2.4 μL Dharmafect1 (Fisher, UK) with 237.6 μL OptiMEM (Fisher, UK) and incubating at room temperature for 5 min. Meanwhile 12 μL of 5 μM siRNA was mixed with 228 μL OptiMEM. Diluted Dharmafect1 was then mixed with the diluted siRNA and incubated at room temperature for 30 min. The cell medium (in the 6-well plate) was replaced with 1920 μL of compete medium. The transfection mixture (480 μL) was then added to each well, giving a 25 nM final siRNA concentration. The cells were incubated in their transfection medium at 37°C, 5% CO2 for 48 hr before further experimentation.
SKBR3 cells (300,000) were seeded in 35 mm glass- bottomed imaging dishes (MatTek) and incubated for 24 hr in complete medium. The transfection mixture was prepared the following day by mixing: transfection reagent 2.4 μL Dharmafect1 (Fisher, UK) with 237.6 μL OptiMEM (Fisher, UK) and incubating at room temperature for 5 min. Meanwhile 12 μL of 5 μM siRNA was mixed with 228 μL OptiMEM. Diluted Dharmafect1 was then mixed with the diluted siRNA and incubated at room temperature for 30 min. The cell medium (in the 6-well plate) was replaced with 1920 μL of compete medium. The transfection mixture (480 μL) was then added to each well, giving a 25 nM final siRNA concentration. The cells were incubated in their transfection medium at 37°C, 5% CO2 for 48 hr before further experimentation.
4
Optimized Immunofluorescence Imaging of Focal Adhesions
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Hs578T cells forming prominent FAs were grown in DMEM supplemented with 10% FBS at 5% CO2 and 37°C. Before imaging, cells were replated onto fibronectin-coated (20 µg/ml fibronectin) glass-bottomed imaging dishes (MatTek Corporation) for 4 h at a density of 5,000 cells/cm2 to avoid ECM remodeling. Cells were washed twice in PBS before fixation and permeabilization for 10 min in 2% paraformaldehyde and 0.1% Triton X-100, an optimized fixation procedure to preserve adhesion structures, followed by three washes with PBS and blocking at 4°C in 0.5% BSA (or goat serum for antitalin staining) overnight. Primary abs were diluted in PBS with 0.5% BSA and then incubated simultaneously for 30 min at 4°C and for 1 h at RT. Samples were washed five times with PBS, incubated for 1 h with secondary ab diluted in PBS plus 0.5% BSA, and washed five times with PBS. To obtain optimal staining, saturating ab concentrations were determined empirically by titration. For STORM experiments, the following ab concentrations were used: 9EG7 (1 µg/ml), 12G10 (40 µg/ml), and AIIB2 (1 µg/ml). For STED and confocal microscopy, the following ab concentrations were used: K20 (50 µg/ml), 12G10 (40 µg/ml), 9EG7 (8 µg/ml), Huts-4 (250 µg/ml), AIIB2 (6 µg/ml), mAb13 (25 µg/ml), antivinculin (33 µg/ml), antitalin (178 µg/ml), and anti–kindlin-2 (26 µg/ml).
5
Culturing Mesendoderm Explants on Fibronectin
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Glass-bottomed imaging dishes (Mattek) were coated with human fibronectin protein (1 mg/mL stock solution diluted 1:40 in PBS) by pipetting the solution onto the dish and incubation at 37°C for 1–3 hr. Fibronectin solution was removed, and the dish allowed to dry for 15 min at 37°C. Explants of the HH4 or HH8 MSP region were prepared as follows. Wild-type embryos were removed from the egg, washed in PBS, and the MSP region isolated using a micro-dissecting knife. Explants were placed on the fibronectin coated dish and oriented mesendoderm (ventral) side down using an eyelash tool and cultured in GMEM (Gibco) + 10% foetal bovine serum (FBS) + 1% Pen/Strep.
6
Live-Cell Imaging of GFP-Rab10 in Hepatocytes
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Hep3B or HuH-7 hepatocytes transfected with GFP-Rab10, as described above, were plated on glass-bottomed imaging dishes (MatTek Corporation) and starved for 1 hour in HBSS + Ca2+/Mg2+. The cells were then immediately imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging) at 37°C and 5% CO2. Images were acquired every 20 s. Movies were converted from 320-pixel × 240-pixel TIF image sequences in Adobe Photoshop CC using H.264 encoding.
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