SH-SYS5 cell culture for dummies...
Posted 12 October 2009 - 02:19 PM
I am quite new into the cell culture world (just know the very basic) and I need to use SH-SY5Y to do some overexpression and silencing experiments followed by protein and RNA extractions or imaging so I have a lot of (probably very stupid) questions:
- what should I use to grow those cells: flasks, dishes?... Any laminin or other coating?
- what is the most appropriate medium, DMEM/F12 seemes to be the most used but I saw a lot of difference in the concentration of serum, glutamate... Do you have any recommended brand?
- what is the best plating density for those cells, how fast are they growing?
- to passage them they said on the cover sheet that both the cells in suspension and attached cells should be used but I have no idea of what is the best speed to spin the cells in suspension, any suggestion?
- do you have any suggestion for transfection protocols to use either for overexpression or with siRNA, any thoughts about the Dharmacon Accell system?
Posted 12 October 2009 - 03:55 PM
2) check the conditions that the cells are supplied with - have a look at the ATCC website and see what they recommend. Use he medium the cells came in initially, but you can wean them on to other mediums if necessary. 10% serum is probably what you want to aim for. Invitrogen (Gibco) is probably the biggest supplier of media, though Sigma and a few other companies also make them.
3) Plating density depends on the size of the cells, check the conditions on the ATCC website.
4) spin you cells gently, 100 RCF (g) maximum is enough for all cells that I can think of.
5) For overexpression I use Fugene6 (Roche), for RNAi I use Lipofectamine RNAiMax (Invitrogen), follow the protocols supplied with each. Dharmacon is pretty good too, they should be fine.
Posted 14 October 2009 - 10:57 AM
I have just received my cells and will play with them.
If you do not see me coming back on the forum it means that everything is going well...
Posted 21 October 2009 - 01:00 PM
My cells are growing, so far so good.
When you are doing overexpression or siRNA, are you using all the cells or only the one that are adherent?
Posted 21 October 2009 - 03:49 PM