Team:Debrecen-Hungary/protocols/ Transfection

Transfection protocol with PEI nuclear receptor construct (for COS1 cells)

Scientific Background

The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.

Overview

Performing the protocol from beginning to end takes approximately one and a half day and it is divided into two major procedures:

1.Plating COS1 cells into 48 well plates in order to get 60% confluency of adherent cells for transfection

The goal is to get 60% confluency of adhaerent cells on a 48 well plate before transfect them

2. The PEI mediated transfection itself

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

Materials

I.Plating COS1 cells:

48 well plates

DMEM Medium containing 10% FBS

Trypsin-EDTA

1x PBS

Brüker-chamber

centrifuge tubes

serological pipettes

pipettes and tips

vacuum aspirator

Pasteur pipettes

Sterile laminar air flow box

incubators

II. PEI mediated transfection

Plated cells

Serum Free DMEM Medium

sterile 100 mM PEI solution

sterile 150 mM NaCl solution

TE –buffer (filtered)

Plasmids: Beta-Gal (normalizer), Luciferase (tracer), Receptor1, Receptor2, VDR-1 (MOCK, negative controll) - their concentration has been measured previously

Eppendorf tubes

centrifuge tubes

serological pipettes

pipettes and tips

vacuum aspirator

Pasteur pipettes

Sterile laminar air flow box

incubators

Procedure

I. Plating COS1 cells:

1. Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 37°C

2. Get the cells to the sterile box in a closed cell culturing flask or Petri dish.
You should open it only under the sterile box.

3. Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette

4. Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette

5. Incubate the Cos1 cells for 3-5 mins with 2 ml of trypsin-EDTA at 370C,
then check them under phase-contrast microscope whether they are detached from the surface

6. Add 4 ml medium(you can change the dilution level depending on the cell number, in order to be able to count the cells easier) to the trypsinised cells,
re-suspend them and check them under phase-contrast microscope to make sure that you got individual cells

7. Prepare the Bürker-chamber and do a cell count

8.To reach 60% confluency the day after plating, we put 21.000 cells into each well

For a 48 well plate, if we calculate with 60 wells, we put into a 50 ml centrifuge tube: - 21000x60= 1.320.000 cells [ cellnumber in 1 ml / 1.320.000 ML cell susp. ] , and if the total volume of the wells are 200 ul, - we fill the cell suspension up to 200x60 ul= 12 ml with 10% FBS DMEM

9. By using a repeating pipet, put 200 ul from this suspension into each well, swirl the plate

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

II.PEI mediated transfection

1.Prepare the sterile box, and prewarm the medium up to 37°C

2.Change the medium at least 1 hour before transfection to Serum Free DMEM.
(gently remove used medium from the cells using vacuum aspirator with Pasteur pipette, then pipette 200 µl Serum Free DMEM

3. Mix gently plasmid solutions (Do not vortex!)

4. Dilute plasmids to 0,1 µg/µl cc. with TE-buffer in sterile Eppendorf tubes

5. Prepare DNA mixes:

T A B L E I N S E R T R E Q U I R E D
Prepare the mastermix: 129,6 ul bGAL, 165,6 ul Luc

Put 66-66 ul Mastermix 1 into four Eppendorf tubes. The plasmids of special receptors have to be added to each Eppendorf tubes (7 µl from each)

in the case of PPAR gamma construct transfection:
B-gal: 180 ng/well
Luc: 230 ng/well
GAL4-PPARg: 120 ng/well
as we need 25 µl 150 mM NaCl solution/well,
we put 16x25=400 ul into each DNA mix Eppendor tube, then vortex briefly

6. Prepare PEI mix in 4 pieces of eppendorf tubes:

A. Per 1 well:
PEI μL 1.3
NaCL μL 25

B. Per 16 well:
PEI μL 20.8
NaCL μL 400

vortex the PEI- NaCl mixes in the Eppendorf tubes briefly.

7. Add PEI mixes to DNA mixes dopwise , then vortex briefly. (Do not add DNA mixes to PEI mix!)

8. Incubate transfection mixes for 20 mins at room temperature

9. Add total transf.mix.volume in one epp.tube/16 µl transfection mix to each well in drops, then swirl gently the plates

48-well plates, 8 rows, 6 columns:

1st row: (VDR-)x2

2nd row: Rec1+ VDR-

3rd row: Rec2+VDR-

4th row: Rec1+Rec2

5th row:(VDR-)x2

6th row:Rec1+ VDR-

7th row:Rec2+VDR-

8th row:Rec1+Rec2

10. Incubate the cells for 5-6 hours at 370C, 5% CO2

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

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