SH-SY5Y Immortalized cell lines; researching neurotoxicity? - (Mar/18/2013 )
I am new to cell culturing. Although I have read, read, and read about cell cultures, I am still confused about the many cell cultures/protocols that are available. For Alzheimer's Disease, I have seen that SH-SY5Y immortalized cell lines are used. How does this work?
I am currently interested in researching microglia-neuron interactions when a specific protein is present. I specifically want to see the effects of microglia (neurotoxins) when the protein is present. What co-culture would work best? This may be too vague. How do you know if you can add a fluorescent indicator to a co-culture?
Any tips on preventing contamination (dots and other contamination) would be great.
Thanks for the responses!
SH-SY5Y is a neural like cell line derived from a neuroblastoma patient, if that is any help to you. I'm not sure what you mean by "how does this work"
I can't comment on co-culture of neurons or microglia, never having worked with them. What sort of fluorescent indicator were you thinking of adding? There are many protocols out there for transfecting cells with DNA that can produce GFP tagged proteins or doing immunofluorescence (after testing). There are some live uptake dyes too. Check out the Molecular probes handbook if you can, this will give you some idea of the things you can and can't do (and the scale of what is available).
Contamination is a topic that deserves a whole book of its own, the best thing you can do is practise your sterile technique as much as possible (and/or learn from an experienced cell culture person). The use of antibiotics in cell culture is becoming more and more frowned upon as it is now known to hide (but not remove) sub-visible contaminations, and teach people to be slack with their sterile technique. Routine testing for mycoplasma should also be carried out to ensure that your cells are not contaminated, as these bacteria can cause a whole heap of effects in the cells that may alter your experimental results. Most journals now require that you show that your lines are not contaminated at the time of paper submission!
Regular scheduled cleaning of the incubators, hoods and waterbaths will help lower the incidence of contaminations. The use of gloves and closed-front lab coats (change regularly) is also a key factor in preventing contamination. Spray everything down with 70% ethanol or methanol (cheap bulk ethanol is fine, it doesn't have to be Analar or anything) before it goes into the hood and spraying your gloves each time you go to put them into the hood is also a big help. If you can, keep a dedicated set of pipettes only for cell culture. It should also go without saying, but a commonly missed thing is having micropipette tips that are only opened when in the hood (i.e. not also used on the open bench).
Global Moderators, thanks! Can immunofluorescence, after testing, also be performed with a C. elegans model? Is it possible to use two different dyes in one culture?
I don't know if your model would be transferable to C. elegans at all, you would have to test it for yourself. It is definitely possible to use more than one dye in culture, I think there are up to about 30 different fluorescent colours you can potentially use, which have been used in neural studies before. It depends a bit on what you want to do and how you want to do it. If you just want to do IF, then you can do this with fixed cells and antibodies or you could do it with live cells and do something like FRET (fluorescence resonance energy transfer).