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Upstate - Procedures and Protocols - Transient Transfection of Cos-1 Cells
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Upstate Cell Signaling Symposium
The first annual Upstate Cell Signalling Symposium will take place in Dundee, Scotland (UK) from 6 - 9th June 2004 and is entitled:

Regulation and Therapeutic Potential of the PI 3-kinase/PKB Signalling Pathway.

The symposium is co-sponsored by Sir Philip Cohen and Prof. Dario Alessi (The MRC Protein Phosphorylation Unit) in conjunction with Upstate.

Participation is strictly limited to 300 delegates, so early registration is recommended.

Transient Transfection of Cos-1 Cells

Note: This protocol has been optimized for Cos-1 cells. Successful transfection of each cell type requires optimization of the basic protocol. Variables to consider for optimization include, but are not limited to, cell density, transfection reagent, duration of transfection, and DNA concentration.

1. Seed a six-well tissue culture plate with 1-2 x 105 Cos-1 cells in 2ml of DMEM supplemented with 10% fetal calf serum. Incubate for 18-24 hours at 37C in a CO2 humidified incubator or until the cells are 40-60% confluent.

2. Dilute 1-2 micrograms of DNA into 100 microliters serum free DMEM in a sterile tube. To this tube, add 6 microliters of PLUS Reagent found in the LIPOFECTAMINETM PLUS Reagent kit (Life Technologies). Allow mixture to stand for 15 minutes at room temperature. In a second tube dilute 4 microliters of LIPOFECTAMINETM Reagent into 100 microliters serum free DMEM.

3. Combine the two solutions, mixing gently and incubate at room temperature for 10-15 minutes.

4. Wash cells once with 2 ml serum free DMEM.

5. For each transfection, add 0.8 ml serum-free medium to each tube containing the LIPOFECTAMINETM PLUS Reagent - DNA complexes. Mix gently and overlay the complexes onto cells.

6. Incubate the cells for 3 hours at 37C in a CO2 humidified incubator.

7. Remove the DNA-containing medium and replace with 2 ml of DMEM supplemented with 10% fetal calf serum. Incubate cells at 37C in a CO2 humidified incubator for an additional 48-72 hours.

8. Assay cell extracts for gene expression or activity at 48-72 hours post-transfection.

LIPOFECTAMINETM PLUS is a registered trademark of Life Technologies, Inc.

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Preparation of Modifed Radioimmunoprecipitation (RIPA) Buffer

Activation of Sodium Orthovanadate

Preparation of Cell Lysate

Western Immunoblotting of Proteins

Troubleshooting Tips for Western Immunoblotting

Immunoprecipitation of Proteins

Immunoprecipitation / Kinase Assay

Troubleshooting Tips for Immunoprecipitation / Kinase Assays

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)

Phospholipid Vesicle Preparation

Chromatin Immunoprecipitation (ChIPs) Assay Kit

Forskolin Treatment of Cells

Preparation of Brain Cytosol

Immunohistochemistry with Fixed Paraffin-Embedded Tissue Sections

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

Immunohistochemical Methods to Detect Nitrotyrosine

Immunocytochemistry with Adherent Cells

Differentiation of Stages in the Cell Cycle

Elution of GST-Fusion Protein from Glutathione Agarose

Purification of Antiserum or Ascites by Protein A/G Chromatography

Molecular Weights of Some Common Proteins

Transient Transfection of Cos-1 Cells

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