Hi everyone
I am performing experimental metastasis assays using B16F10, LLC or MCA205, injecting the cells via the tail vein, and the number of lung nodules is quite variable.
Is there any tips to lower this variation: best syringe /needles ? injection speed ? new needle / syringe for each mouse ? cell preparation ?
I am injecting 100ul of cell suspension with 0.5 CC syringes, 28G 1/2 needles (BD). I usually use the same syringe /needle for 5 mice, refilling the syringe between each mice.
I inject at most 20 mice per batch of cell suspension (60-90min), cells are kept one ice.
Cells are cultured by trypsinization, passed on a 40um strainer, washed in PBS, and resuspended at the right concentration in PBS.
Thanks in advance
Your protocol seems fine. If you're right in tail vein injections it should be ok. The following are possibilities
1. There could be variations in mice immune system even though they are littermates.
2. The cells might have low number of metastatic cells so presence of metastatic cells within this 100ul matters.
3. Try to avoid straining with filters (which might prevent temporary clumps formed by metastatic cells from being injected). You can disrupt the clumping just before injection either by pipetting or by tapping (preceded by proper trypsinization). (if you have large volume of cells aliquot them so that pipetting in between every mice injections will not damage the cells.
4. The cells you use may not be efficient in metastasis.
5. Try a sub-cuteaneous injection to see if it can really metastasize (or an orthotopic injection will do).
be sure mice are 8-12 weeks of age. if younger or older then they are in the growth phase of their hair cycle and have a lot more circulating growth factors from the keratinocytes--this causes variability because each mouse cycles differently.
Hi Bertrand
I agree with the replies above that the inherent character of individual mice can affect the degree of metastasis. Also, the handling of cells matters a lot.
1. How many times you have subcultured the cells? Using early cultures of tumor cells helps. Also, you can isolate tumor cells from already established subcutaneous tumors and use them for metastatic experiment. It would certainly increase the metastatic efficiency.
2. the time duration between start of cell preparation and injection should be kept minimum possible.
3. Use HBSS instead of PBS. It works better.
4. There is no need to use strainer. Cell clumping can be prevented by pipetting the cell suspension 1-2 times just before injection.
5. After preparing cell suspension, it is better to aliquot the whole suspension in small volumes ... 1 ml. before filling the syringe, pipette 1-2 times to resuspend well,
5. You said it takes around 60-90 minutes to inject 20 mice. This time duration should be kept minimum. One thing you can try is fill ~5 syringes at a time with cell suspension and inject them. Refill and repeat. It should save you some time. OR you can have a colleague to fill the syringes for you. You can lower the injection duration significantly.
Good Luck!
More you will render your metastatic models "homogeneous" and more artificial will be your "models" as compared to the clinical situation.
Today, 90% of cancer patients still die from their metastases.
The metastatic process is amazingly complex at the biological level.
I stronly encourage you to have an in-depth reading of the articles here attached.
The article with the specialists' direct comments on xenografts is very important.
Do not forget the problem of "metastasis dormancy", which is so difficult to combat in clinics and that somewhere you ask here how to "technically bypass" this biological phenomenon..
Once you will have actually read these articles, I guess that you will have a "new view" about how to handle your "metastattic" models.
Best regards
Robert
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