How to analyze Angiogenesis HUVEC cell tube formation assay using ImageJ Software
Вставка
- Опубліковано 19 вер 2024
- Link to the Angiogenesis analyzer macro
imagej.nih.gov...
Thanks to DR. MOHSINA for helping in making this tutorial
If you have any questions or comments, please feel free to put it in the comments sections below. Thanks
Please subscribe for more ImageJ videos
Thank you so much for this! You literally have saved me so much anguish. and also for a note in the comment section about reducing the size of the image to get the macro to run. Am so very grateful to you.
Thank you very much! I'm delighted to hear you found the video beneficial. It's encouraging to see that the community is also offering useful advice in the comments section. That's what makes networks like UA-cam so effective: we can all learn from one another. If you have any questions or need clarification on anything, please ask.
You are my lifesaver...... thank you....
Thank you so much for watching and finding the video useful! I'm really glad it was helpful for you.
Excellent thank you
You are welcome. I am glad you liked it.
Thank you for this video. I had a question, so you uploaded the pictures you got the stat so now with that what do you infer, I am new to this so I am trying to learn what yo infer from the results and what all should I mainly look for in this case. So that I can also try comparing images too
Hello thanks for reaching out. Here is the original publication for the angiogenesis analyser imagej (Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of “Endothelial Tube Formation Assay” and “Fibrin Bead Assay”) The link is www.nature.com/articles/s41598-020-67289-8 . Let me know if you have more questions.
@nrttaye4033 Thanks for the paper, but it doesn't say tho like what the stats signify .and what is the most important factor among them to look for. If you can explain on that stuff it would be great.
Hello, thank you so much for the video. I have a question, I am trying to count the number of tip cells, which I assumed would be found as the number of extremities in the analysis. But I ran the same program on a blood vessel image which has no extremities but the analysis still came back with over 100 extremities for that image. I am wrong in assuming that tip cells would be equal to extremities or is there another reason that a blood vessel image would show 100s of extremities? thank you
Hello, you are welcome. I am not sure about that. Could you please send me a sample image at nandarajtaye@outlook.com I could troubleshoot it
nice tutorial
Glad you liked it!
Hello. We're planning to do a CAM Assay, what outputs should we focus on if we are looking for the length of blood vessels and the number of branches. Thank you!
Hi. Using this plugin the info for number of branches can be obtained. In this video at 2:19 mins, please check the option "show branches". To perform the vessel length analysis, there are other plugins available. Have a look at these videos that may help in your CAM analysis. ua-cam.com/video/fcqNutzR7F4/v-deo.html ua-cam.com/video/3VZ_Qjvmh64/v-deo.html
Hello last question. How to do 8 bit Rgb?
Hi, to convert an RGB image to 8 bit color, click on image, type and choose 8-bit color. A dialogue box will appear (number of colors 2-256). choose 256 and then click ok.
This link doesn't have the macro code anymore. Where can I find it? can someone send it to me?
hello i agree the link is not working. Here is an alternate link. All the best. imagej.net/ij/macros/toolsets/Angiogenesis%2520Analyzer.txt
Hello what are mostly the parameters if it is a CAM assay experiment? thanks for the video. so much helpful!
Hi thanks for the question. for measuring the growth, vasculature and angiogenesis in an CAM assay the parameters mostly used can be Blood Vessel area, vessel diameter, branch numbers, length and branching patterns. The parameters mentioned in the video at 2 min 11 sec will mostly produce these outputs in the results. The blood vessel area/density and diameters may be quantified using the pixel intensity using ImageJ separately. You may also be interested in this video (ua-cam.com/video/v5URAQI_lOE/v-deo.html). Hope this helps. Let me know if you need for info.
hi. I loved your video and I use thi program. I want to know what does only the yellow lines mean?
Hi, I am glad you liked the video. Thanks for the question. In the video frame 5:14 to 5:25 the yellow line is referred to as master segment. Hope this helps. Let me know if you need more info.
Hello. Thank you so much for the video. I look at fluorescent images and angiogenesis. What parameters should I look for? so far, I do total length but that number seems very large
Hi, I am glad you liked the video. I think you are on the right track looking at the tube length. The units seemed large as these are in pixels (can convert pixels to micrometer). Specific units can be assigned if there is a scale bar in the image. Many of the publications use the parameters 1) No. of tubes 2) Tube area 3) No. of segments, 4) Total segment length and 5) No. of junctions. However it greatly depends on the experimental readout. Additional data can be generated with the advantage of having fluorescent images. Recently I read an article where the tubes were stained with endothelial marker PECAM-1 (to visualize the tubes) and apoptosis marker (active caspase-3) to help in identifying whether apoptotic cells contribute to anti-angiogenic drug treatments (shortening of tubes).
hello, thank you so much for this! I did the HUVEC tube formation assay last week for first time, and I was really confused how to quantify the result. I have one question. The parameter I need is only 'tube length', and in this case, what parameter in this macro should I look for? Total branches length? I'm really confused. Sorry for my bad english skills.
Hello, you are welcome and thanks for reaching out. I appreciate you watching the video. You asked a great question, so don't stress about your English. It is possible to measure the 'tube length' in the HUVEC tube formation assay by looking for the parameter "show branches, show master segments and show suppressed elements" in the angiogenesis analyzer settings. Yes, you might be interested in looking at the "total branch length" in the results table.
Please don't hesitate to ask further questions if you need more information or clarity. I hope your experiments turn out well.
Thank you for kind reply😀 I have one more question then, what does 'total segment length' patameter means?
Hi, Ive followed the instructions but mine came out as min error- not converging. Any Idea how to resolve this?
Hello, an image file of 10 Mb or more size takes a very long time to process or it shows an error. please try reducing the file size under Image, adjust and size and then run the process again. alternatively, try changing the brightness or contrast of the tube formation so that it is very distinct from the background color and run again.
Hello sir. Thank you for your video. Can we use this software to analyse the angiogenesis done through CAM assay. Kindly need your reply sir
Hello, for CAM assay analysis there is another way using Fiji. I will upload the video in a day or two. Please stay tuned till then. Thanks
@@nrttaye4033 Thank you sir. Very kind of you. Waiting for your video sir
@@ajayeswarr6690 here is the link ua-cam.com/video/3VZ_Qjvmh64/v-deo.html. all the best
Hello! When I run the macro I get even things in the background of my picture as networks and branches, not only the actual cell-cell connections. How do I solve this issue? Thanks in advance
Hello. I was in a similar scenario to you once. I believe the macro can detect even minor background/noise. Remove the speckles/dot-like structures with ImageJ functions, or change the contrast such that the background is reduced. I noticed that an uneven brightness in the image might also create this problem at times.
If your image's background is clean and you're still getting, try altering the parameters in the angiogenesis analyzer settings.
Let me know if that works for you.
Thanks! I tried with the speckles function and the contrast but I still have the same problem. I will play around with the angiogenesis analyzer settings and see if it works@@nrttaye4033
Hello, thank you for your video. According to your installation method, the macro is successfully installed, and the icon does appear. However, the analysis is unsuccessful for a long time, it stops at the dilated step, and the analysis has not been completed after waiting for about 30 minutes to 1 hour. I would like to ask if you know of any solution？ thank you
hello, thanks for the question. I encountered this issue with large image file size. A 10 Mb file size image takes me about 3 minutes to complete the process. Smaller the file size faster is the process. Less than 1 Mb takes about 5 seconds. please reduce the file size under Image, adjust and size and then run the process again. Hope this helps you. Let me know if it worked. All the best.
@@nrttaye4033 I adjusted the image file size from 40 Mb to 1 Mb, and it works!!! thank you so much!
The macro is no longer available...
hello I agree the link is not working. Here is an alternate link. All the best. imagej.net/ij/macros/toolsets/Angiogenesis%2520Analyzer.txt