I am currently using a 20uL tip to create scratch but apparently, with every new biological replicate exp, I do not get uniform or similar scratch as the previous exp. Any hacks/tips on how to achieve a uniform scratch every time? Also, Pls recommend the best tool/software to accurately measure the widths? Thank you.
Hello Ankit Naik
I used to do the scratch with a 20μl tip and it is always going well. Here are some tips that may help:
if you do the assay using 6 or 24 well plates, you may use the plate cover as a ruler during scratching.
don't twist the tip and try to fix the angle between the tip and the plate during scratching. it is better to run the tip over the cells once. regarding the second part of your question, ZEN Blue Software is doing well and you can set it up with your inverted microscope camera https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html
you can also use any photoshop program, in this case, the width will be in pixels.
these may help you to optimise your assay
https://www.nature.com/articles/nprot.2007.30
In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro
Article Optimized Scratch Assay for In Vitro Testing of Cell Migrati...
Good Luck
Article An introduction to the wound healing assay using live-cell microscopy
Hi. Some learnings from my repeated failed experiments:
1. Keep your 20ul tip straight while using plate cover as ruler, just dont twist, try to keep the pressure constant.
2. If a little difference occurs, its not a big problem, you can normalize as taking the initial area of scratch as 100% and determine the subsequent observation as percentage of initial scratch.
3. For analysis, install image J, its latest version is ImageJ2, which comes with name Fiji. Then you have to install a plug in
there is an option, where you can either automatically select the wound area or select it manually and calculate the area by this plugin. File attached.Just follow instructions.
All the best
This is very relatable. In addition to the tips above there is also the "cheat" option- some companies have well inserts that you can put in, and then you seed the cells into the insert, remove the central barrier and you will have a nice clean, uniform gap. This also avoids "trauma" to the cells near the wound. The inserts are however more expensive than just doing a scratch yourself. In all honesty some variation is likely to be expected. More repeats, and looking at % gap closure might be more important than the absolute size of the gap in my opinion. Would love to hear other's views.
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