MCF10A nuclei were microinjected with polyethylene glycol (PEG) modified 100 nm red fluorescent Carboxylate-Modified nanoparticles (Invitrogen). We followed established protocols to coat Carboxylate-Modified nanoparticles (Invitrogen) with amine-terminated PEG (42 (link),43 (link)). 10 μl PEGylated particles (3.6×1010 particles/ml) were sonicated for 30mins before loaded to Femtotip II (0.5 μm, Eppendorf). The particles were then microinjected into the nuclei using an Eppendorf InjetMan microjection system. Cells were incubated at least 6 hrs. before imaging. Time lapse images of fluctuating particles embedded in the nucleus were acquired on a Nikon TE2000 inverted epifluorescent microscope with 60x/1.49 NA oil immersion objective (Nikon) and CCD camera (CoolSNAP, HQ; Photometrics). Cells were maintained at 37ºC in 5% CO2. Tracking of particles was achieved by using TrackMate plugin of ImageJ (44 (link)). The trajectories of the particles were then used to calculate time-averaged mean squared displacements using @msdanalyzer Matlab tool (45 (link)).
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Nanoparticle Dynamics in Nucleus
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Corresponding Organization : University of Florida Health
Other organizations : Northwestern University, Dana-Farber Cancer Institute, Harvard University, Stanford University, University of Chicago, Tennessee Technological University, University of Florida
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Variable analysis
independent variables
- Microinjection of PEG modified 100 nm red fluorescent Carboxylate-Modified nanoparticles (Invitrogen) into MCF10A nuclei
- Fluctuation and movement of the nanoparticles embedded in the nucleus
- Time-averaged mean squared displacements of the nanoparticle trajectories
- Incubation time (at least 6 hrs. before imaging)
- Temperature (37ºC)
- Atmosphere (5% CO2)
- Imaging equipment (Nikon TE2000 inverted epifluorescent microscope with 60x/1.49 NA oil immersion objective and CCD camera)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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