Transfection – 18/07/2011 Data collection and analysis – 20/07/11

HeLa Cell Transfection Experiment

96 well plate, 10,000 cells per well

10,000 HeLa cells were seeded in each of the highlighted wells, 24hours before transfection, allowing the cells to properly attach to the plate.

24hours after seeding complexes of DNA and nanoparticle were formed to the following ratios:1. nTMag 0.1ul – GFP 0.1ul2. OzBio 0.1ul – GFP 0.1ul3. nTMag 0.1ul – LUX 0.1ul4. OzBio 0.1ul – LUX 0.1ul

The DNA and nanoparticles were allowed to complex for 15mins at room temperature. After the DNA and nanoparticles had complexed, the cell culture medium was aspirated and 100ul of new medium (-FCS) and nanoparticle/DNA complex were added to the appropriate wells, as outlined in the diagram below:

After the medium and complexes had been added to wells, the plate was placed on a magnefect-nano 96-well oscillating magnet array for 30mins, in an incubator (37ºC,5% CO2). The magnefect-nano system was set at 2Hz frequency, 30min duration and 0.2mm displacement. This process caused the nanoparticle/DNA complexes to be drawn down onto the cells. The oscillation increased the agitation of the nanoparticles onto the surface of the cells, encouraging the cells to take in the nanoparticle/DNA complexes via endocytosis.

The hypothesis was that 48hours later, the cells would have transcribed the DNA and translated it into a protein. In the case of GFP, the cells with the protein would glow green under a fluorescent microscope, and the cells that were transfected with LUX DNA would produce light (luminescence).

After the 30mins on the magnet array, the cells were taken out of the incubator and off the magnet array. The wells were then aspirated of medium and nanoparticles, so as to make sure the transfection occurred over 30mins only. 100ul of new (+FCS) medium was then added to the wells, which were left for 48hours in an incubator at 37º,5% CO2.

nTMag 0.1ul –

GFP DNA 0.1ug

OzBio 0.1ul –

GFP DNA 0.1ug

nTMag 0.1ul –

LUX DNA 0.1ug

OzBio 0.1ul –

LUX DNA 0.1ug

Transfected HeLa cells using nTMag nanoparticles with GFP DNA visualised with fluorescent microscope.

Magnefect-nano plate, 96-well plate, 2Hz, 0.2mm, 30min, seeded 10,000 cells/well

Fluorescent

nTMag 0.1ul – DNA 0.1ug

Light Composite

Transfected HeLa cells using OzBio nanoparticles with GFP DNA visualised with fluorescent microscope.

OzBio 0.1ul – DNA 0.1ug

Fluorescent Light Composite

Magnefect-nano plate, 96-well plate, 2Hz, 0.2mm, 30min, seeded 10,000 cells/well

Luciferase Assay on HeLa cells transfected with LUX DNA using nTMag and OzBio nanoparticles.

Magnefect-nano plate, 96-well plate, 2Hz, 0.2mm, 30min, seeded 10,000 cells/well

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Luminescence Intensity

Well Number

Luciferase Bright-Glo Assay - HeLa cells 48hours

nTMag 0.1ul - DNA0.1ul

OzBio 0.1ul - DNA 0.1ul

Control - Glo lysisbuffer 30ul - Bright-Gloassay reagent 25u - NoCells

From the results of the luciferase assay we can suggest that the nTMag nanoparticles have a higher transfection efficiency with LUX DNA than the OzBio nanoparticles. We can suggest this because the wells transfected with nTMag nanoparticles have consistently higher luminescence than the wells transfected with OzBio nanoparticles.