- Primary cells have a finite life span. They are exactly like the cells that you would find in your own body, so they do die after a certain period of time in culture. The amount of time primary cells survive in culture varies according to cell type.
- Primary cells can be diverse. Cancer cell lines often come from a single patient – for example, HeLa cells came from one woman, Henrietta Lacks. Primary cells, on the other hand, can come from a variety of people. Having a variety of primary cell types from many different donors is especially useful when carrying out early drug testing— it’s important to ensure that the drug is effective for everyone. Before testing the drug in human patients, researchers can use primary cells from different donors to verify that the same effects are observed.
- Primary cells can change in culture. For this reason, and because primary cells do not live forever, Lifeline Cell Technology strongly suggests carrying out experiments on primary cells using earlier passages.
- Primary cells have not been modified in any way. Except for the enzymatic and/or physical dissociation required for extracting the cells from their tissue of origin, primary cells are not altered in any way. Immortalized cell lines, on the other hand, are often transformed either with cancer genes, viruses or other inducible modifications, which could alter the outcome of experiments.
- Primary cells are finicky. Cell lines are hardy, mostly because they have to last a long time in culture, and be capable of surviving multiple rounds of cryopreservation and thawing. Primary cells, on the other hand, tend to be more difficult to maintain in culture.
GFP Cell Preparation
· GFP-Labeled Primary Cells employing Amaxa nucleofector transfection methods are detached from the culture flask and immediately cryo-preserved in vials. Each vial contains 0.5-1.0x106 cells per ml and is delivered frozen. GFP marked primary cells are verified by a fluorescent microscope.
· Cells can be seeded on 30-60 mm culture dishes ready for experiments (or cells may be expanded for 1-2 passages at a split ratio of 1:2) under the cell culture conditions specified by Cell Biologics. Repeated freezing and thawing of cells is not recommended.
Protocols for GFP-labeled Primary mammalian cells
· Harvest the cells by trypsinization. Count an aliquot of the trypsinized cells and determine cell density.
· Centrifuge the required number of cells (0.5-1.5 x106 cells per sample) at 220xg for 5 minutes at room temperature.
· Resuspend the cell pellet carefully in 100 µl room temperature Nucleofector™ Solution per sample.
· Combine 100 µl of cell suspension with 5-10 µg pmaxGFP™ Vector (recommended for initial optimization).
· Transfer cell/DNA suspension into certified cuvette; sample must cover the bottom of the cuvette without air bubbles. Close the cuvette with the cap.
· Select the appropriate Nucleofector™ Program.
· Insert the cuvette with cell/DNA suspension into the Nucleofector™ Cuvette Holder and apply the selected program.
· Take the cuvette out of the holder once the program is finished.
· Add 500 µl of the pre-equilibrated culture media to the cuvette and gently transfer the sample immediately into the culture dish. Use the supplied pipettes and avoid repeated aspiration of the sample.
· Incubate the cells in a humidified 37°C/5% CO2 incubator until analysis. GFP expression is often detectable after only 4 – 8 hours but ideally, cells should be left undisturbed for 24 hours. GFP expression efficiency depends on different cell types.
Transfection efficiencies: ~40-60% by using Amaxa's Nucleofector device