Team:Debrecen-Hungary/protocols/ Transfection

FuGENE 6-nuclear receptor transfection of COS1 cells

Scientific Background

FuGENE 6 transfection reagent is a multi-component lipid reagent that forms a complex with the DNA, then transports it into animal cells. FuGENE 6 transfection is used as standard method in many different laboratories due to its simple methodology, low cytotoxicity, and ability to provide high transfection efficiency even in the presence of serum.

Overview

The transfection is divided into two main steps: 1. plating COS1 cells into glass cell culture vessel 2. the transfection itself

1. Plating COS1 cells into glass cell culture vessel:

The aim is to get cells of 50-80% confluency in the chosen vessel for the day of transfection.

Required materials:

- COS1 cells in a T75 flask (regularly passaged, proliferating well - best in the log-growth phase)
- Trypsin/EDTA solution
- 1 x PBS solution
- 10% FBS containing DMEM medium
- LAB-TEK glass chamber slide with 8 chambers
- 15 ml centrifuge tube, tube holders
- Bürker chamber, pipettor, serological pipettes, pipettes, pipet tips, Pasteur pipettes
- sterile laminar air flow box, 37°C incubator, 37°C waterbath
- 70% ethanol, kimwipes

Steps:

1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, wipe the box with 70% alcohol.

2. Prewarm the DMEM medium, Trypsin/EDTA and the 1x PBS in the 37°C waterbath (10-15 minutes)

3. Take the DMEM, Trypsin-EDTA and PBS and squirt the tubes and bottles with alcohol beforeyou put them in the sterile box. Put the pipettor and the tube holder into the box
(after you sprayed them down with 70% ethanol), and load the serlogical pipettes directly,without spraying down into the box.

4. Spray hands with ethanol. Remove the flask from the incubator and quickly place in hood. Fire-sterilize the neck of the
flask. (Do not spray flasks with ethanol).

5. Attach a Pasteur pipette to vacuum,turn on vacuum system by opening vacuum valve in hood. You should fire-sterilize the end of the pipette,after this step do not touch anything outside the flask. Aspirate the used medium from the cells by touching the bottom-side corner of the flask with the Pasteur-pipette.

6. Washing step:Add 2-3 mL of 1x PBS to flask by using pipettor and a serological pipette (Release the PBS onto the side of the flask, do not push the solution out directly onto the cells because they can come up easily). Lightly swirl PBS on base of the flask. Aspirate PBS from flasks by using a Pasteur pipette and vacuum.

7. Add 2 mL trypsin-EDTA to Flask. You can release the solution directly onto the cells, from now it does not matter if they come up. Lightly swish trypsin.

8. Place flask in 37°C incubator until detached (3-5 minutes for COS1 cells, depending on the temperature of the Trypsin- opt. temp: 37°C)

9. Remove cells from incubator. Tap side of the flask on hard surface of your hand. Repeat several times. Visually check to ensure lumps of cells are dispersed.

10. Check cells under phase-contrast microscope to confirm that cells are detached from the surface.

11. Put the falsk back to the sterile box, add 4 ml of 10% FBS containing DMEM medium to dilute trypsin (you can change the dilution level depending on the cell number, in order to be able to count the cells easier). Medium contains antitrypsin.(Note: The liquid suspension now contains the cells.)

12. Carefully resuspend cells by using pipettor and serological pipettes. You can repeat this step until you get individual floating cells (microscope check needed).Put the cells into a 50 ml tube, for easier handling.

13. Prepare the Bürker chamber and do a cell counting:

- Cell counting in a Bürker chamber:

The Bürker chamber is the most commonly used tool for counting cells. Now comes a short introduction about how to use a Bürker chamber:

• The Bürker chamber from outside:
  

* How to use the Bürker chamber:

1) At first, clean the chamber with alcohol and water, put the thin glass slide onto the thicker slide,
compress them together by using the metal screw.

2) Resuspend the cell suspension (for even distribution) in which you want to do the cell counting.

3) Take 10 ul from the cell suspension (using sterile tip),

inject into the chamber(marked with arrow) by touching the border of the two glasses with the pipet tip.

4) Place the chamber under the phase-contrast microscope and try to find the lines.

- count inside 3 big squares (an example is on the next picture):

 then we take the average of the 3 big squares, and it will give us the number of the cells in 0,1 ul

 we take it 10 in the factor of 4 times and it will give us the cell number in 1 ml cell suspension.

NOTE: Don't forget that the cell suspenson in the chamber is now not sterile,
don't put it back into the cell solution. Clean the chamber with alcohol and water.

1. To reach the appropriate confluency the day after plating, we put 50.000 cells into each well.
For an 8 well chamber, if we calculate with 12 wells (because the volume loss), we put into a 15 ml centrifuge tube:
- 50.000 x 12 = 600.000 cells [ in milliliter: counted cell number in 1 ml / 600.000 cells ]
- we fill the cell suspension up to 8 x 300 ul = 2, 5 ml with 10% FBS DMEM
(total volume of the wells are 300 ul)

2. With a 1 ml pipette put 300 ul from this suspension into each well. Sometimes invert the cell suspension containing tube
(the cells decent). After you finished the plate, swirl it circularly.

3. Incubate the cells for 1 day at 37°C, 5% CO2.

2. FuGENE 6 transfection:

The goal is to introduce foreign plasmid DNA into the plated COS1 cells 24 hours after the plating.

Materials required:
- Plated cells in a LAB-TEK 8 well glass chamber slide ( with 50-80% confluency)
- FBS/antibiotics/other additives Free DMEM medium
- Sterile FuGENE 6 transfection reagent in a tightly capped glass vial
- Plasmids on ice, in known concentrations: Beta-Gal (normalizer plasmid), Luciferase (tracer), Nuclear receptor, VDR- as
negative control, the plasmids are solved in sterile TE-buffer
- 1,5 ml sterile Eppendorf tube, pipettes and tips
- Sterile laminar air flow box, 37°C incubator, 37°C waterbath
- 70% ethanol squirt bottle, kimwipes

Steps:

1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, spray down the base of the box with 70% ethanol, and wipe down with kimwipes.

2. Spray down your hands, the DMEM medium, the glass Reagent vial and the pipettes and tips with 70% ethanol. Put these and the plasmids and the eppendorf tube into the sterile box.

3. Transfection mix reconstitution:

  3.1 Invert the room temp. FuGEGE 6 Transfection Reagent glass vial 2-3 times to distribute the components.
  3.2 Dilute the FuGENE reagent with Serume/antibiotics/other additives free DMEM medium – the order and manner of   addition is critical:
     Label a 1,5 ml eppendorf tube.
     Pipet 75 ul FBS/antibiotics/other additives Free DMEM into the eppendorf tube.
     Pipet 6 ul FuGENE 6 Transfection Reagent directly into the medium, without allowing contact between the plastic    wall and the undiluted reagent.
  3.3 Vortex the mix for 1 second.
  3.4 Incubate the mix for 5 minutes at room temperature.
  3.5 Add 500 ng from each of the three plasmids (receptor, Luciferase, beta – Gal.) into the diluted FuGENE 6   transfection reagent.
  3.6 Vortex the Transfection Reagent:Plasmid mixture for 1 second.
  3.7 Incubate the mixture for 20 minutes at room temperature.

4. Remove glass chamber slide with plated cells from the incubator, place in the sterile box without spraying down with ethanol.

5. Note: We don’t need to remove the culturing medium (10% FBS containing DMEM) from the cells, it does not have any effect on
the transfection efficiency.
Add 9 ul Transfection mix in a dropwise manner to each well. Swirl the chamber slide to ensure distribution over the entire surface.

6. Put on the cap of the slide chamber. Return the cells to the 37°C incubator until the assay for gene expression is to be
performed.
Note: it is not necessary to remove and replace the transfection mixture-containing medium with fresh medium until the assay, only if you used FBS Free medium during the whole experiment (to avoid the cell starvation).

7. Clean up after yourself, place the FuGENE reagent to +2 - +8 °C and be sure if the cap is tightly turned on the Reagent.

NOTES:

- store the reagent at +2 - +8, with the lid very tightly closed, in the original glass vial.
- Do not allow the reagent to contact plastic walls (pipet directly into serum free medium) to keep the maximal biological activity.
- Do not use siliconized pipet tips and tubes.
- To prepare transfection complexes for larger experiments or parallel experiments, proportionally increase the quantity
according to the total surface area of the cell culture vessel being used. (ul FuGENE Reagent: ug DNA = 4:1, the used vessel in
this case has a 79,21 cm2 surface area)

Notes & troubleshooting

References

1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2].

2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511

Links

[http://www.youtube.com/user/debrecenigem2010#p/u/13/r8DlyOCRJRs Video I]