you will need to repeat the experiment other wise your result will not be significant.

technically we perform the scratch using 10 microliter. the analysis is done by measuring 6-12 points in the scratch. you can present the data as area remaining open at the time point or % of closed area

hello

normally for wound healing assay u need  the wound created to be quiet similar (not necessary the same). the distance between the wound influences the rate of closure. yet still u can analyse your result by following this software called tscratch, which automatically does the calculation for you. it uses the initial wound created and wound closure during the expt to give you a measurement of how your treatment varies from the control. please find attached the protocol. for the pipette tip some use 10 ul and others use 200 ul  or even 1ml tips. but this depends on how fast ur cells migrate to wounded area. eg is leukocyte cells they migrate very fast so when u use 10 ul tip, the wound might be close quiet fast. 

cheers sis!!!!!!!!!!!

http://www.biotechniques.com/multimedia/archive/00045/BTN_A_000113083_O_45150a.pdf

http://www.nature.com/nprot/journal/v2/n2/full/nprot.2007.30.html

Hi Brunette, I have done many migration assays through the years and personally I think that the tissue culture wound healing assay is by far the hardest one to do and interpret.

To me it sounds like the miRNA treated cells are sticking together more strongly that the control cells. When you do the  wounding on the control cells, you are ablating individual cells. When you do the wounding on cells that stick tightly  together you are just dislodging a part of the cell sheath that will resettle quickly and close the gap.

This migration assay is supposed to count individual cells crossing into the wounded area, but if the cells are migrating as a sheath, then they will migrate slower that individual cells

Thanks all for the grate answers .

I think i will subtract the 48 h gap size from the initial gap size to give me the gap closure rate . I hope this is right.

Dear Khamal , 

I did use the T.scrach software for measure the gap area.

The question is how to measure the closing rate (migration rate) !

For miRNA treated cells , the wound close slower than the negative control which fact faster.

I did lots of wound healing assays using live cell imaging and taking pictures at every hour for 24 hours (depending on cell type you can do it longer or shorter).For calculating the velocity rate of migrating cells I use an ImageJ Plugin. In Fiji this is already implemented in the plugin tab :Plugins --> tracking --> Manual tracking. I track around 25 cells to get enough data. Then you can save this data and open the Chemotaxis and migration tool from Ibidi, open your saved data and enter some values (like time interval, and the distance in um. The tool calculates the individual cell velocity as well as the directionality of the tracked cells, plus some other parameters like euclidean distance and accumulated distance. (it's free, http://ibidi.com/software/chemotaxis_and_migration_tool/?xe456c=d6e87fbc185a36ebaf7f6b6e3bba35f2) 

Hi, for wound healing assay it is not important what is the size of wound area among the groups at 0h. The most important thing is that you document the 0h and 24h field should be same so that when u use software like t-scratch to quantitate it gives u the migration oh the same field. So the most important thing is to focus the same field at 0h and 24 h for each group, then u take the extent of migration in each group and evaluate the differences among the grouos . repeat it 3-4 times as independent experiment for stats.

In principle, considering individual 0h time point-area  as 100% and tracking the percentage closure with time is OK. But if your scratch size differ so highly (If I understand correctly - 38% and 70%) between the test and control, you may have to reproduce the data to be completely sure. How many scratches/wells have you measured?. On the other hand a big control scratch healing better than a small test-treated scratch could indicate a strong phenotype. Good Luck..

One year later I can hopefully ask a related question- to Dafna, when you are using the chemotaxis plugin for Fiji, how do you plot multiple tracks? I am only able to plot single tracks and I can't figure out why.

Hi Claire, I am really sorry...I never got a notice that you posted a message. Were you able to figure it out in the mean time? If not let me know and I can help you (it has been a while since using the chemotaxis plug-in :P)