Cell Culture and Transfections

Cell Culture

Cells express the membrane receptors which are investigated as potential targets for therapeutic reagents in early stage drug-discovery.

For saturation and competition radioligand binding assays, cells can be used either live or after membrane harvesting, to determine affinity and/or IC₅₀ of test compounds. For cellular uptake and release assays, live cells are used to determine IC₅₀ and EC₅₀, respectively.

We have experience of culturing both cell line adherent cells (e.g. Hek-293) and cell suspensions (e.g. THP-1), as well as primary cells.

Use in Live Cell Assays

Cells are maintained in T175 flasks incubated at 37°C, 5% CO₂ in their specified growth media. They are passaged at confluency and media is changed when required. Aliquots are cryopreserved in usable aliquots and stored at -152°C. For assays they are seeded into 24-well plates and grown to confluency overnight. Cells are washed with PBS and used directly in an assay (e.g. live cell uptake, competition or saturation radioligand binding assays).

Use for Membrane Preparation

Cells are cultured in T175 flasks (as above) until confluent when membranes are harvested. Flasks are washed and cells are removed. An isotonic solution is used to break cells apart and release the cell membranes, which are subsequently washed using centrifugation. Protein concentration is determined, and membranes are stored as usable aliquots in a suitable storage buffer at -80°C. On the day of an assay aliquots are defrosted on ice and used to provide the membrane target in a radioligand binding assay.

Transfection

Overexpressed membrane proteins can be used in either live cell or membrane based radioligand binding assays.

Cloning and DNA Amplification

DNA is first cloned into a Plasmid, a vector containing antibiotic resistance. The plasmid can then be amplified using a bacterial expression system. Bacteria cells (e.g. Ecoli DH5α) containing the plasmid are cultured under selective pressure (using antibiotic resistance). A larger amount of DNA can then be extracted using an extraction kit (E.g. quigen maxiprep). This can then be used in the transfection process.

Non-Viral Transfection

Transfection can take place using a variety of methods. We standardly use lipid nanoparticle technology (e.g. lipofectamine).

The plasmid (containing the DNA sequence of interest) is taken up into vesicles which are able to merge with mammalian cell membranes and deliver this plasmid into the host cell. Cell culture media is changed and cells are cultured until use in a live cell assay or until the membranes are harvested.

Immortalised cell lines can be used to overexpress membrane proteins. The process involves introducing DNA using a vector, often with an antibiotic resistance gene into a cell line. The cells are then cultured in the presence of the antibiotic to create selective pressure. The cells undergo the process of transcription and translation and the membrane protein is expressed.