Team:Debrecen-Hungary/protocols/ Media PEI Preparation

Preparing medias for eukaryotic cell culturing, preparing PEI solution for transfection

Scientific Background

I. The Media

Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line.

II. PEI solution

PEI is used for PEI-mediated transfection, we need a 10 mM solution of it.

Overview

I. The Media

For culturing cells and for ligand treatment we use DMEM medium completed with 10% FBS, 1% Penicillin-streptomycin and 2% L-Glutamine

For transfection we use DMEM medium without FBS, but other constitutives are included

The preparation procedure takes 40-50 minutes including the FBS melting time

II. Preparing PEI solution

The reconstitution consists of 3 steps including dissolving PEI, adjusting pH and sterilization by filter sterilizer

Materials

I. For the Media:

500 ml Basic DMEM ( Dulbecco’s modified Eagle Medium ) ordered from Sigma (D5671)

50 ml FBS (Foetal Bovine Serum)

10 ml 200 mM L-Glutamine ordered from Sigma(G7513)

5 ml 100x Penicillin-Streptomycin ordered from Sigma(P4333)

Serological pipettes, Pipettor

Sterile laminar flow box, kimwipes, 70% ethanol sprinkle bottle

II. Preparing PEI solution:

Pipette, pipet tip, glass, analitical scale

MQ water (double distilled, sterilized)

MW=25.000 Pei (ALDRICH 408727)

15, 50 ml tubes

pH measuring electrodes

HCl-solution

Sterile filter – with 0,2 um diameter pores

Procedure

I. Preparing Media:

1. Prepare the laminar flow box: turn on the ventillation, wait for 15 minutes, clean the bottom and the glass of the hood with 70% alcohol, and wipe it with kimwipes.

2. Put the Basic DMEM solution, the FBS, Penicillin Streptomycin and the L-Glutamine solutions into the 37°C waterbath and wait for 30 minutes (The FBS is melting slowly)

3. Take out the melted solutions from the waterbath, wipe them, squeeze them down with 70% ethanol and load them into the hood as well as the serological pipettes and the Pipettor (squeezed down)

4. Put these amounts into the basic DMEM by using a pipettor and serological pipettes:

A. For 10% FBS medium:

Basic DMEM: 500 μL

FB: 50 μL

L-Glutamine: 10 μL

Penicillin-Streptomycin: 5 μL

B. For Serum free medium:

Basic DMEM: 500 μL

FB: -

L-Glutamine: 10 μL

Penicillin-Streptomycin: 5 μL

5. Invert the bottle, mark it with your name, actual date and with the constituents

6. Put the bottle to 4°C, clean up after yourself.

II. Preparing PEI solution:

1. Dissolve 4,5 mg pure PEI in 8 ml MQ water, mix well (maybe it takes one day for proper dissolvation)

2. Neutralize the solution with HCl. The final pH should be between pH 6,5-7,5

3. Adjust the volume to 10 ml

4. Filter sterilize through 0,2 um pores

5. Store the solution at 4°C

Notes & troubleshooting

References

1. Yves Durocher, Sylvie Perret, and Amine Kamen,"High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells". [http://nar.oxfordjournals.org/content/30/2/e9.full Nucleic Acids Res. 2002 January 15]; 30(2)

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