Techniques for tumor xenograft establishment - (Oct/16/2013 )
Recently I attempted a tumor innoculation using MDA-MB-468 cells combined with Matrigel , the suspension was a 1:1 volume to volume mixture, and the final suspension had 2X10^6 cells in the total 200 uL volume injected.
I injected the dual flanks subcutaneously , and once I penetrated into the SC I made a small pocket with the syringe, then while slowly injecting the cells I moved the needle to the left and right in a fan motion to spread the cells.
Now, it has been 2 weeks since the innoculation, and you can see a very small lumps that are very spread out.
Is it better to inject it directly as one mass without moving the syringe needle? Also, should I have put much more cells because the growth is really small and limited?
What do you guys recommend, can somebody give me some tips? Was it such a small volume that was dispersed?
I was thinking to inject again using 200uL of the 1:1 mixture with matrigel with a total of 5X10^6 cells directly as once lump?
When you guys penetrate the SC and form a pocket do you inject all the cells together as a lump, or do you sway the needle left and right to deposit the cells over the entire suface to increase the surface area?
I have had really bad luck with this tumor induction and would really appreciate some tips from you guys!
Hi I think you are doing quite right. The only thing that I don't like is to move the needle in a fan motion to spread the cells. This is unnecessary. Once the needle has penetrated the skin to SC space, push the needle a short distance further in the SC space (so that the cells won't easily come out once you withdraw the needle) and inject your cells to let them form a visible lump. If you spread the cells, it is harder for them to grow into visible tumor.
In you case, I think you can wait for another week to see if tumors appear. If not, you can increase the number of cells to 3-5 million. Beware if you reinject cells to the same location you may get very nasty tumors (maybe the matrigel previously injected can potentiate cell growth and invasion.)
Hello I have a question
I am planning to induce tumors on 30 mice on the dual flanks for a total of 60 tumors. I am prepare the cell suspension and matrigel mix for 70 tumors just to have some extra on hand. After i form the cell suspension and matrigel suspension, do i load all 70 syringes at the same time in increments and store these on ice, and then inject the mice one by one?
I am trying to minimize the variation by doing this as a big experiment in one batch, is this the proper technique?
I am assuming the loaded syringes will have to sit at least 1 hour or so while I am loading all the syringes, what do you think is the best way to go about doing this?
Should the 30 mice be inoculated at the same time or seperated into 15 each and done after another so the cells are not sitting too long in syringe.
whether to divide the mice to batches depends on how you are experienced and the time needed to finish 30 mice. For me, I can do the 60 injection in 20 min and feel there is no need to do it in two different times. I don't pre-load the syringe with cells. I use one syringe (1 cc) to take 1000 ul cells each time and inject 5 sites (200 ul each) while keep the tube containing cells on ice. After finishing the 5 sites, I use the same syringe to take another 1000 ul. I change to a new syringe if I have to inject a different cell line.
Thanks alot buddy! Are you using a 1:1 mixture with matrigel?
I know they have different formulations of the matrigel, are you using the high protein content or the normal version of matrigel?
I believe the protein content can vary from 10mg/mL to around 5mg/mL or so after reconstitution in media
We use the normal version of matrigel and mix it with cells at 1:1 ratio (volume).