Discussion of all aspects of cellular structure, physiology and communication.
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So, I'm a chemist who does immunology research. I've always had an immunology collaborator, so I've never had to learn tissue culture technique. She is leaving soon though to take on another position, and so, if I want to continue my research, I need to learn the technique.
She's willing to train me, so I won't be completely on my own. However, I was wondering if anyone could recommend any tissue culture text books that might be useful for me to get? I just feel like it'd be good to read about the techniques before I start doing them.
In addition, our current studies are done in stably transfected B-cells, but our research is focused on dendritic cell proteins. It's getting to the point where we need to start working with DCs instead of a model cell line. I know there aren't any immortal DC lines, so most people just induce monocytes. How feasible would it be to generate moDCs with limited tissue culture experience? Is it considered a very advanced technique? Our lab doesn't have this technique yet, so I'd be most likely learning to do it on my own.
Monocyte-derived DCs should be easy enough to culture even with limited experience.
Basically you need cell-culture plates that allow monocytes to stick onto its surface (they attach readily to most "hard" plastic surfaces), serum-free culture medium (such as AIM-V), growth factors (poly-I:C, GM-CSF) and an incubator.
Toss freshly isolated PBMCs on a properly sized plate (e.g. 24-well plate) in AIM-V, incubate for, say, 2 hours at +37C and wash the wells "firmly but gently" several times to remove non-adherent cells (i.e. lymphocytes). Add fresh medium and something like 100 ng/ml GM-CSF and incubate for 24 hours. Then add 25 ug/ml of poly-I:C and incubate another 24 hours, and voilá! With some luck, you have just generated dendritic cells!
You can the harvest them with a cell scraper if you so wish, but if you intend to stimulate other cells with them, maybe just leave them on the plate and add the to-be-stimulated cells to the well along with antigen (or alternatively first pulse the DCs with antigen for 4 hours in +37C and wash). The cells are stuck quite firmly to the plate, so harvesting may break some of them. Use of EDTA or some other such ion-binding substance may help removing the cells alive.
Use a microscope to make sure you do not have contaminating cells on the plate when you start and also to see the morphological changes in the monocytes as they mature. If you have an access to a cytometer, use of antibodies for something like CD14, CD83 and CD86 (if I recall correctly) helps to evaluate the degree of maturation of your cells.
Disclaimer: the above is written based on what I remember of my past protocols, so some errors may exist. Check the study design from immunology journals to ensure you get things right.
3 posts • Page 1 of 1
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