Application Note 07

Protocol for Cell Transfection in ibidi µ-Slides

Due to the broad spectrum of transfection methods, this application note only
describes an example of DNA transfection conducted in µ-Slides VI 0.4. Any othertransfection protocol may be adapted in a similar way to the handling in ibidi µ-Slides.
The transfection reagent Metafectene µ FluoR applied in this protocol provides thepossibility of a direct tracking of the reagent uptake by the cells. It is a lipid labeledwith a fluorescence tag (rhodamine) which can be detected by fluorescencemicroscopy.
Principle:
In the first step the plasmid attaches to the lipid which results in a so called lipoplex.
Subsequently the lipoplex is added to the cell suspension where it is taken up byendocytosis. In the cell lumen the DNA is released from the lipoplex and is taken up
into the cell nucleus at the next cell division.

ibidi µ-Slide VI 0.4

Cells transfected with GFP in µ-Slide VI 0.4

Material
Cells:
Medium:Buffer:Transfection reagent:
Plasmid:
Reaction tubes:
Slides:HepG2
DMEM 10% FCS
PBS (1x)
Metafectene µ FluoR (Biontex Laboratories GmbH)
pCMV-GFP (0.5 mg/ml)
polypropylene reaction tubesµ-Slide VI 0.4, ibiTreat_____________________________________________________________________________________________________
Application Note 07© ibidi GmbH, Version 2.0, Helga Wagner, January 11, 2010
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Application Note 07
Preparations one day in advance:- Put the slides overnight in the incubator to ensure the plastic is prewarmed
and the gas is exchanged entirely.
- Equilibrate the medium for the cells in the same way. Place a small vessel
into the incubator and leave the cap slightly ajar to make leak the excesspressure.
Preparation of transfection solution:- Warm up transfection reagent, plasmid solution and PBS buffer to room
temperature.
- Dilute 2 µl of the transfection reagent (Metafectene µ FluoR) in 36 µl 1x PBSand mix by gently pipetting up and down.
- Add 4 µl of your plasmid solution to the reagents dilution and mix gently by
pipetting up and down.
- For constitution of the lipoplex allow the mixture to stand for 15 minutes atroom temeprature. In the meantime prepare the cell suspension.

Preparation of cell suspension:- Detach and count your cells as usual. Adjust a cell suspension of 4 x 105
cells/ml in DMEM. For filling six channels of the µ-Slide VI prepare 200 µl ofcell suspension.
Transfection:- Add the lipoplex (42 µl) solution the cell suspension (200 µl) and again mix
gently with the pipet.
- Fill 30 µl of this mixture into each of the channels and incubate the cells forfive hours at 37°C and 5% CO2. Make sure that evaporation is minimized.
Eventually put the µ-Slide into an extra Petri dish with a water soaked wipe.
- During incubation time you can observe the lipoplex uptake by live cell
imaging on an inverted microscope.
- After five hours check your cells on a microscope. They should have adheredwell.
- Exchange the medium with fresh medium: Put 120 µl of fresh medium into
one of the channels reservoirs. The medium will flush through the channel tothe opposite reservoir. Aspirate 120 µl with a pipet from the oppositereservoir. Avoid aspirating the medium directly from the channel’s inlet!
- Add 120 µl fresh medium to the first reservoir and observe your cells.
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Application Note 07
Observation of the transfected cells
At this point of the protocol the cells have taken up the lipoplex with the plasmidDNA. For a successful transfection the lipoplex is set free from the endocytotic
vesicle and is transported into the cell nucleus. This mostly happens during cell
division when the nuclear envelope breaks down.
For observation with time lapse microscopy put the slide on an inverted microscope
equipped with an onstage incubation system (e.g. ibidi heating system). Take apicture every 10 minutes. Use a filter set suitable for GFP to visualize the expressionof the transfected DNA and a filter set for rhodamine to trace the arrangement oflipoplexes.
In addition to the fluorescence recording you may also record phase contrast
pictures. You will see how cells are dividing and afterwards start to express GFP.
Phase Contrast

Rhodamine-labelled
Lipoplex

GFP

Overlay

HepG2 cell line transfected with GFP: The transfection reagent is labelled with rhodamine.
It is visible as small red conglomerates in the cell lumen. Transfected cells produce GFP,
which is diffused in the whole cytosol, glowing green.
Pictures were taken with a 20x objective on a Nikon TiEclipse, 24 hours after adding thelipoplex to the cells.

Watch the movie on www.ibidi.com !
_____________________________________________________________________________________________________Application Note 07© ibidi GmbH, Version 2.0, Helga Wagner, January 11, 2010
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Application Note 07
Factors of impact
Concerning cell transfection, there is no principal difference between a µ-Slide and astandard vessel like e.g. a 6 well plate. Hence, all rules for best transfection results
apply for both substrates. Nevertheless, we would like to give you some hints
derived from our own experience.
Once you have chosen the best transfection method for your cells, transfection
efficiencies may still vary from 0% to near 100%, which depends on different
experimental factors:a) Reagent concentration: Since all transfection reagents are more or less toxic,
you have to find out which is the maximum concentration that is supported byyour cells without a significant mortality rate (should be < 10%). This is very
often the most important parameter.
b) Incubation time: If cell mortality is a problem, try to incubate the cells theshortest possible time the reagent's manual recommends. If you find low
yields in healthy cells, extend the incubation time to the maximum.
c) Plasmid concentration: There is a connection between plasmid concentrationand transfection efficiency. However, this parameter's impact is usually low
as long as you follow standard protocols.
d) Preincubation time for Plasmid-Reagent complex formation: Onlyrecommended as a fine-tune parameter. Usually you are best advised when
you follow the average times from the reagent's manual.
e) Plasmid purity: a crucial parameter for transfection success. However,
sufficient purities are easily achieved with all common plasmid purification
kits. Therefore, this is usually not a source of trouble.

_____________________________________________________________________________________________________Application Note 07© ibidi GmbH, Version 2.0, Helga Wagner, January 11, 2010
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