I am attempting to determine if loss of my protein of interest decreases migration of NIH 3T3 cells. Any tips would be awesome!
What should I coat my dishes with? I have tried using Poly-Lysine and Fibronectin. My cells are detaching, I have tried to plate various starting amounts of cells but I haven't been able to figure out the right amount to yield a monolayer.
Take a look at the attached method from SciGine related to a wound healing assay utilizing 3T3 cells. You need to start with a monolayer of cells, and, in this case, they used PFA to help the cells attach + migrate into the wound.
http://scigine.com/method.php?ID=31695
You can simply coat with gelatin. It is efficient in my experience. Good luck
If your cells are of good quality no coating is needed, you just have to work with good cell density (low cell density causes stress). When you perform scratches coating agent is removed, at least partially. This will cause variability and artifacts. Moreover, these cells behave differently on different types of coating – meaning that coating itself may cause artifacts related to cell migration.
I recommend you to start from relatedly high cell number – seed such amount that will give you sub confluent plates within 24 h. After that do scratches and make pictures for analysis every 24h (do not forget time zero before treatment, just after you did scratches). For proper analysis of migratory capacity you should prepare two sets of replicates – one in regular conditions and another with inhibited proliferation. For inhibition of cell division you may use Mitomycin C which is commonly used to generate mitotically inactive cells in culture.
With regard to scratches manual, in each plate we do two parallel horizontal lines and two parallel vertical lines. This gives you from each plate 12 roads and 4 junctions to take pictures from. Try to mark areas (on the bottom) where you take pictures, and every day take the same frames. In such a way, even if you work with triplicates only, you will have enough pictures for digital measuring and for proper statistical analysis (Anova, t-test and Cohen's d).
Good luck!
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