As long as the cells are in exponential growth when you collect them from culture, are Mycoplasma free, you confirm that your cell viability is high, and they don't sit around for an hour or more before you get them into the mice then you should be able to get close to 100% take. If you're getting much lower than that, check the origin of the cell line (STR sequencing) and try seeding in cell culture medium rather than PBS. You shouldn't need to resort to matrigel with 231s, they are pretty robust and should be highly tumorigenic.

Thank you so very much for your answer. Our infrastructure is very limited right now so do you think I should treat the cell culture with anti mycoplasma antibiotics for 3 weeks before injection (the ones that dont affect cell growth) and the medium that I resuspend to inject should it be complete med or dmem only (I just dont know whether FBS is ok to inject) Thanks a billion again

Media containing FBS is usually OK to inject into immunedeficient mice. If you cant check directly for Mycoplasma contamination (there are kits you can buy), then treatment with a standard antibiotic regimen for these bugs shouldnt do any harm.

very recently we used the same cell lines(MD-MBA-231) for making breast cancer xenograft (we used matrigel) the detailed procedure I'm attaching here, you can have a look, and we tried different dilutions of cell lines 3 million to 5 million cell lines per mice.

MD-MDA231 cell-line xenograft:

MD-MBA-231 cell lines from ATCC were revived, allowed for 2 passages, from the second passage, based on the requirement of cell lines and the number of mice available for making xenograft, the cells were grown in 10 no. of 100 mm cell culture grade dishes, (usually one 100mm dish contains 8.8X106 cells), once cells were 80% confluent, cells were harvested and counted using an automated cell counter (Biogenix) by Typhan blue exclusion method and cross-checked by counting manually also. We obtained 35x106 cells/ ml from all 10 dishes.

1. Mice have injected 2x 106 to 5x106 cells for tumor development, in-detailed-

Once cells were harvested and counted by using Typhan blue method using an automated cell counter, we got 10x106 cells/ ml, per each mouse we injected 100 µL,2x 106 cells in 20% Matrigel (Corning, NY USA) (80 µL cells+ 20 µL Matrigel).

Cell-lines based xenograft was done according to our requirement

Confirmation: the appearance of a small bleb after, injection, indicated successful cell line transplantation.

Observation:4-6 weeks for tumor development.

Robert Clarke thank you so very much I shall try it with complete medium tomorrow

Kirankumar Nalla thank you so very much! we inject 3 million cells in PBS but in some mice after a week, the bleb disappears. is it something you guys observe as well? Thank youuu

Halin Bareke, we too observed, one or two mice have no blebs like appearance on the site where we injected cells for tumor formation. but almost after 4-5 weeks tumors started appearing gud enough to go measuring.

here I'm attaching the protocol reference we followed for this.

link: https://tcr.amegroups.com/article/view/24543

paper title: Exploring protocol for breast cancer xenograft model using endothelial colony-forming cells

Kirankumar Nalla Thank you so very much. we kept seeing tumor formation and then we lose them. It has been 4 weeks and we even did repeat injections in some and the tumors spontaneously regress:( I have read many papers on this but we seem to be stuck in our model :( I am gonna increase the number of cells ...can you think of any other refinement? thankss a lot!

Poor tumor and/or slow tumors growth take can be an indication of Mycoplasma contamination. 231s should normally grow easily and fairly quickly. Sometimes, the cells settle in the tube as you go from mouse to mouse and the inoculum size can vary unintentionally (so some mice may not get enough cells). You dont need to agitate the tubes that much to keep the cells in suspension but they will also start clumping in the tube if you wait too long to get them into the mice (and that can have the same effect)...Bob

I agree with Robert Clarke, maybe cells might be settled down in tubes by the time you're using them for injection. I use to carry an icebox and had taken help from seniors to avoid this problem.

Hello once again, my problem still persists :( despite I have done the following

1. checked for mycoplasma contamination

2. increased the number of injected cells to 6 million

3. made trials with matrigel

I am thinking may be establishing tumors in CD1 nude is more challenging than Balb/c nude. does anyone have any experience on that? Thanks a million for helping me out in this dire situation