SH-SY5Y
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SH-SY5Y cells can form mounds of undifferentiated cells, which then spread differentiated cells into the surrounding area. This growth formation can be referred to as the "over-easy formation"
History
The cell line SH-SY5Y is a third generation neuroblastoma, cloned from SH-SY5, which is from SH-SY, which is from SK-N-SH. The original cell line was isolated from a woman's metastatic bone tumor in 1970. In each successive generation, the nucleus was removed and put into a new cytoplasm in the process known as cloning. This particular generation is four years old and has a blood type A positive.
Morphology
The cells possess an abnormal chromosone 1, where there is an additional copy of a 1q segment and is referred to trisomy 1q. SH-SY5Y cells are known to be dopamine beta hydroxylase active, acetylcholinergic, glutamatergic and adenosinergic. The cells have very different growth phases, outlined in the surrounding pictures. The cells both propogate via mitosis and differentiate by extending neurites to the surrounding area. While dividing, the cells can look so different from the differentiated cells, that naif scientists often mistake on or another for contamination. The dividing cells can form clusters of cells which are reminders of their cancerous nature, but certain treatments such as retinoic acid and BDNF can force the cells to dendrify and differentiate.
Media and Cultivation
The most common growing cocktail used is a 1:1 mixture of DMEM and Ham's F12 medium and 10% supplemental fetal bovine serum. The DMEM usually contains 1.5g/L sodium bicarbonate, 2mM L-Glutamine, 1mM sodium pyruvate and 0.1 mM nonessential amino acids. The cells are always grown at 37 degrees Celsius with 95% air and 5% carbon dioxide. It is advised to cultivate the cells in flasks which are coated for cell culture adhesion, this will aid in differention and dendrification of the hybridoma.
Splitting
Splitting is the act of taking a cell rich culture and dividing it up into many less dense cultures. This is done either for preventing overcrowding, or for expanding the number of cultivated flasks. Although every lab does this differently, the general procedure is as follows.
- Aspirate off the old cell media
- Rinse twice with sterile PBS
- Add about 2 milliliters of Trypsin with 0.53mM EDTA: a protease and a metal chelator, respectively
- *This will break apart all of the cellular proteins that adhere the cells to the flask.
- **If the trypsin is left too long, the cells will fall apart, but not long enough and the cells won't seed very well.
- **For SH-SY5Y cells, the best time is around two to three minutes.
- * Tap, and rock by hand such that all of the cells are covered with trypsin
- * Be sure that the cells begin to flow with the liquid with each rocking.
- Add an approproate amount of fresh, prewarmed feeding media (5mL, 3mL).
- *Note: It doesn't matter, but just know that it must be a set amount, so that it can be further diluted to the correct ratio. The optimum ratio is around 1:30, but 1:50, 1:200, and 1:1000 have all been used.
- *Note: The DMEM has protease inhibitors, which neutralize the actions of the Trypsin.
- Take pipette into the cell-rich media and tritrate gently.
- * Do not tritrate too hard, or the cells will not survive, but not enough and the cells growth will be chunky (it is better to err on the chunky side).
- * When finished, the flakes and clumps of cells should be mostly gone and the media should resemble something like cloudy grapefruit juice.
- Take one milliliter and place into a sterile centrifuge tube. Dilute as needed, (29mL works well).
- Seed or take the diluted cell media and place into a fresh, sterile flask, or dish, or plate.
- Redilute the original flask as needed (or not, newer flasks grow better)
- Place on cap and place flask/dish/plate into incubator
- Pat self on back
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