In one of the experiments using an endothelial tube formation assay, I can see a very clear difference in that the treated vs control group showed differential tube formation in terms of number, thickness, size etc.
Now, I would like to have that data in the form of numbers so they can be plotted on a scale.
Any ideas or methods that help me in quantification of tube numbers, size, thickness etc, or overall tube formation would be helpful.
Thanks.
Dr AmJad Husain, you can assessed the effect of any compound on HUVECs by observing the HUVEC cells under an inverted phase contrast microscope, and take the photographs using a digital camera. The digitised images can then be processed, and analyse using ImagePro Plus software. Tube formation can be quantify by determining the total tube length in four randomly chosen fields.
Hoping it will be useful for you.
All the best in your angiogenesis experiment.
The tubulogenesis literature is varied as to what parameters matter most or are most relevant. Many publications report only the tubule length, but there are many other parameters to consider. My advice would be to perform cell-tracker or immunocytochemistry for the experiment, perform epifluorescence or confocal laser scanning microscopy, and to use an algorithm to skeletonize your images. There is free software available, such as Image J, and MATLAB codes such as RAVE, which can skeletonize your images for you. Once you have a skeleton (which is essentially simplifying your endothelial cells into a stick diagram), I would recommend using one of the above software packages to measure the total tubule length, density of branch points per area, tubule length per area, and a metric that describes the complexity of the network (lacunarity or fractal dimension). See the literature below for a better description of the available tools for measuring these different parameters in different biologically relevant contexts. As with most quantitative measures, one metric is usually not sufficient to describe a biological system, so I would recommend at least reporting a few of the ones listed above in order to capture the complexity of the process.
Gould et al. Microcirculation 2011
Guidolin et al. Microvascular Research 2004.
Dear Amjad Husain ,
you can count the different structures of tubologenesis following the protocol that you can find attached.
"Define visual patterns by looking or photographing the cells at 40X – 200X
magnification at set time at 37°C after seeding on ECMatrixTM (end-point
assay). Assign a numerical value to each pattern. This way a numerical
value is associated with a degree of angiogenesis progression....etc".
It's possible to study the thickness of the capillary-like structures using a specific plug-in of Image J software.
Using tube length and branch point quantification as mentioned above is the way to go. One thing to be cognizant about is that the commercially available "angiogenesis assays" do not take into account that there will be network formation of endothelial cells and then regression over time. So at a specific time point you may see a difference, but that could be because the treatment group had its peak of network formation at a time point earlier than you assayed and the networks have regressed or vice versa. the other issue is are your cell actually forming tubes or are you seeing cell processes that are interconnecting? One would have to section and visualize the tubes to be certain.
Good luck with your experiments.
I second Vivek's opinion. The tube formation changes with time. Usually I will check them every one or two hours. Also the cell density will affect your results. High density will not form tubes but form sheet of cells. Low density will not be able to form tubes. You need to do some test run.
Thank you All. Vivek, Ru-Jeng, David, Annarita, Umar Madhulika, Harish for all good suggestions. Although I could open the tube images in ImageJ but I am still trying to figure out how to measure? I've already done the tube formation assay and have nice pictures already. Any thoughts ?
I would recommend to you run the Matrigel assay and you can do the measurement as you need using image J ( NIH free software). Please take a look to my publication in PNAS and you will understand the method.
Article S-nitrosoglutathione reductase (GSNOR) enhances vasculogenes...
Hi Amjad-
I use the angiogenesis subprogram within Metamorph software. It lets you set the parameters to exclude the nodes, and will do tube length, width, branch patterns, etc from your pictures. It is not free, but it cuts the analysis time down considerably, while giving you data from multiple parameters at once. Good luck.
dear, i'm agree with carol, unfortunetly it's money and time consuming process using Metamorph software. i've recently discover a new informatic platform that allow rapid analysis with multiparameter results (tube length, cell area, nets, tube size....). 5 minutes analysis is needed to receive results, you just sent your picture in the plateform. it's not realy expensive, and before to buy analysis you can analyze 10 pictures for free. before, i've used Metamorph or Image-J, now i prefer this analysis process from IBIDI compagny (Wimasis image analysis platform ).
best regards
I also use the Wimasis Image analysis from IBIDI now. I tried to do it manually with ImageJ, but since I have to analyse over 200 photo's, it really is time consuming. The Wimasis software gave similar results compared with the manual counting, so I switched to that. And it gives you several parameters like total tube length, number of tubes, average tube length, number of loops etc.
Probably too late, but have tried AngioTool, it seems to measure all the possible parameters, provided you have a clean image to start with. It is only available on windows and it is open source.
Dear collegues,
Doing some research on google of "how to evaluate tube formation with Image J" I found these helpul ansewers in here. I hope someone will read this even after all this time has passed. My problem is that Image J count segments were there are cells monolayers, so my control, which has less segments formed (clearly by the image attached) has a higher number of segments (as well the other parameters measured by it). I could exclude them by the width of the segment, I guess, but I don´t know which analysis software allows me to do that... Could anyone help me?
Thank you
Hi you can use wimasis image analysis.
sign up. First time free.
https://www.wimasis.com/en/
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue