For time-lapse phase-contrast and fluorescent microscopy, HeLa cells were plated on 35 mm glass dishes (MatTek) and put in an open chamber (Life Imaging) equilibrated in 5% CO2 and maintained at 37 °C. Time-lapse sequences were recorded every 5 or 10 min for 24 or 72 h using a Nikon Eclipse Ti (more details in section Immunofluorescence) inverted microscope with a × 20 0.45NAPlan FluorELWD or × 60 1.4 NA PL-APO VC objective controlled by Metamorph software (Universal Imaging).
35 mm glass dishes
Manufactured by MatTek
Sourced in United States
The 35-mm glass dishes are laboratory equipment used for cell culture applications. They provide a contained environment for the growth and maintenance of cells. The dishes are made of glass and have a diameter of 35 millimeters.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
Laminin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Metamorph 6, by Universal Imaging (1 mentions)
Fluoview fv10i liv confocal microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Dmem with high glucose, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ti e fluorescence microscope, by Nikon (1 mentions)
7 protocols using 35 mm glass dishes
1
Time-Lapse Microscopy of HeLa Cell Dynamics
2
Chemotaxis Assay of 19F-labeled DCs
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DC motility towards chemokine following labeling with 19F nanoparticles was determined using an agarose spot assay, as previously described66 (link). Briefly, a 0.5% agarose solution was prepared, cooled to 40°C and mixed with chemokine (2000 ng/ml CCL21) or PBS (as –ve control); thereafter the agarose solution was pipetted as 10 μl spots onto 35-mm glass dishes (MatTek Corporation, Ashland, MA, USA). Following harvesting, DCs labeled with 19F nanoparticles were introduced, together with culture medium, to the glass dishes and incubated for 4 h at 37°C (5% CO2). The number of cells entering the agarose spot was determined by acquiring and fusing the microscope images for all the fields of views (FOV) making up the 10 μl agarose spot and counting all cells entering the corresponding spot. Image processing (including FOV fusion) and analysis (mainly cell counting) were done with Fiji (Image JA v1.47p, Open source software, NIH, Bethesda, Maryland, USA)67 (link). For fusing all images the Stitching plugin was used and for counting the number of cells within the spot a semi-automatic procedure using the 3D Objects Counter analyze tool was used.
3
Short-term Culture of Isolated Cells
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Immediately after isolation, cells were placed in a short-term culture (24 h), as previously described [17 (link)]. The cells were seeded onto 35 mm glass dishes (MatTek Corporation, Ashland, MA, USA) coated with 25 µg/mL laminin (Sigma Aldrich, St. Louis, MO, USA) dissolved in PBS (in PBS + 1% penicillin-streptomycin (PS), no Ca2+ and no Mg2+, Sigma Aldrich), in a serum-enriched culture medium composed of M199, 5% fetal bovine serum (FBS, Gibco), 2% PS, and 0.1% 2, 3-butanedione monoxime (BDM). BDM was washed out prior to the experiments.
4
Time-lapse Microscopy of Cytokinetic Abscission
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Two-dimensional cultures (Supplementary Movie 4 and Supplementary Fig. 4a ): For time-lapse phase-contrast microscopy, transient or stably transfected MDCK cells were plated on 35-mm glass dishes (MatTek) and put in an open chamber (Life Imaging) equilibrated in 5% CO2 and maintained at 37 °C. Time-lapse sequences were recorded every 5 min for 48 h (Supplementary Fig. 2a ) or every second (Supplementary Movie 4 ) using a Nikon Eclipse Ti inverted microscope with a × 20 0.45 NA Plan FluorELWD objective (Supplementary Fig. 4a ) or a × 100 1.4 NA PL-APO objective (Supplementary Movie 4 ) controlled by Metamorph 6.1 software (Universal Imaging). This microscope was equipped with a cooled CCD camera (HQ2; Roper Scientific). In Supplementary Fig. 4a , cytokinetic abscission time was quantified in mCherry-tubulin-transfected cells by time-lapse microscopy starting from furrow ingression until the cut of the intracellular bridge. In all, 100 cells per condition were analysed. Supplementary Movie 4 was deconvolved using Huygens Professional software SVI (2 iterations, signal/noise 10).
5
Quantifying Nanoparticle Cellular Internalization
6
3T3-L1 Cell Differentiation Protocol
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3T3-L1 cells were grown in a monolayer tissue culture flask maintained in a growth medium (DMEM with high-glucose, 4500 mg l−1; Life Technologies, Stockholm, Sweden). The medium was supplemented with 10% of heat-inactivated fetal bovine serum, 1% penicillin and streptomycin and 1% non-essential amino acids. The cells were placed in an incubator at 37°C with a humidified atmosphere of 95% oxygen and 5% carbon dioxide. After 2 or 3 days, when the cells were ∼70% confluent, they were split and resuspended in either 35 mm diameter plastic dishes (Nunc), 35 mm glass dishes (MatTek) or 12-well plates at a density of 1.5 × 105 cells/ml, ready to be differentiated when a 90–100% confluence was obtained (∼2 days after seeding the cells). The first differentiation cocktail (D1) contained insulin (Sigma 170 µM), dexamethasone (Sigma; 10 mM) and 3-isobutyl-1-methylxanthine (Sigma, 5 mM). After 2 days, the medium was changed for a second differentiation cocktail (D2) containing 170 µM insulin only. The cells were used days 8 and 9 after the start of differentiation.
7
Calcium Imaging of Transfected Cells
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Transfected cells were seeded on 35 mm glass dishes (MatTek Corporation, Ashland, MA, USA) and incubated with calcium indicator fluo-4AM (5 μmol/L; Invitrogen, Eugene, OR, USA) for 20 min at 37 °C. Next, the cells were rinsed with Hanks' balanced salt solution, and the medium and petri dish were replaced. A closed and thermally controlled (37 °C) stage-top incubator (Tokai Hit Co., Shizuoka-ken, Japan) was inserted over an inverted Nikon TiE fluorescence microscope (Nikon Instruments, Tokyo, Japan) electric bench equipped with a × 60 oil-immersed optical lens (CFI PlanFluor, NA 1.43; Nikon Instruments) and NIS Elements software. A Lumencor diode-pumped light engine (SpectraX; Lumencor Inc., Beaverton, OR, USA), ET-GFP filters (Chroma Technology Corporation, Bellows Falls, VT, USA) and ORCA-Flash 4.0 sCMOS cameras (Hamamatsu Corporation, Bridgewater, NJ, USA) were used for comprehensive testing. Data was collected every 3 min within 30 min.
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