It is inherent in cell culture protocols to modify the media according to what you want to do, when you want to do it. For plating or cell transfers, it’s standard to use chelating agents (e.g. EDTA/Versene) to bind the Ca++ and Mg++ that inactivate trypsin and that are requisite for multiple types of enzyme activity, such as phagocytosis, clotting, and cell adhesion (the integrins needed for attachment to substrate require Ca++). Because this effect is severe, it is not intended to leave cells in CMF (calcium-magnesium-free) solutions for long.

As Ashfaq stated, for injection you want the cells well dispersed. Once in situ the cations will be readily restored, but if you try to inject sticky clumps, many of the cells will never make it through the needle. Simple test: stain the syringes of CM+ and CMF injections with trypan blue and see what is left: I doubt you will be happy. (Note: naïve syringes, never used before, will be charged to attract cells—coat with a flush of l-lysine first).

It is helpful to read a background text on cell culture to understand the chemistry of the cells you’re manipulating—what they can and can’t (or won’t) do is so dependent on their environment, and understanding the hours it may take for biochemical recovery from a suspension medium may be critical to your results. Be sensitive to that term CMF: many cell culture media labels may say only that to describe the modification.

We routinely performed B16-F10 in vivo metastasis assay (tail vein injetcion) and we have always resuspended the cells in regular PBS before injection. Cells are always highly invasive. In our case, the variability often comes with the injection procedure, and also differences can occur depending on the mice strain. Hope it helps!

Thank you, Miguel.

We found that if suspended the cells in PBS, the cell viability decreased sharply after about 2hrs-experimental procedure. That's why we changed to the HBSS, which maintain the cell viability quick well.

The most difficulty we have now is the high variability, which makes our goal to evaluate the drugs’ efficacy impossible. What do you mean “the variability often comes with the injection procedure”?

We use C57/BL6 mouse.

Hi Aiken,

It´s true that we were performing the injections in batches of 10-15 mice, so we never went over 2h with a round of injections. However, we had some issues with some mice strains in terms of making the tail vein visible, or working with too young animals. Best results were obtained in animals around 8 weeks.

Good luck!!

Thank you Miguel, thank you Ashfaq, your response are of great help.

I would like to know what kind of parameter you use to characterize the metastasis? And how about the precision? I prefer to weight the lung organ, since the counting of the cancer clone is difficult in most cases. However, the standard deviation (SD) is as high as equal to the average. this makes evaluation of the drugs' efficacy impossible.

Dear Aiken

do you know the paper: A. Nasulewicz, J. Wietrzyk, F. I. Wolf, S. Dzimira, J. Madej, J. A. M. Maier, Y. Rayssiguier, A. Mazur; A. Opolski, (2004) Magnesium Deficiency Inhibits Primary Tumor Growth But Favors Metastasis in mice. Biochim. Biophys. Acta (Molecular Basis of Disease), 1739(1): 26-32. In this study it is the serum magnesium content that affects metastatic process. As you can see metastatic foci were evaluated by microscopic analysis of the lung. I am surprised that the lack of Ca and Mg disturbs the cell suspension! I would expect the opposite. In addition, some cells do not like to stay in PBS w/o cations for a long time.

Hi Federica, thank you for your information, it's of greate help. It's realy a good story in the paper you provided. I wonder if the magnesium repletion could promote the metastasis in addition to increase the tumor growth.

It is inherent in cell culture protocols to modify the media according to what you want to do, when you want to do it. For plating or cell transfers, it’s standard to use chelating agents (e.g. EDTA/Versene) to bind the Ca++ and Mg++ that inactivate trypsin and that are requisite for multiple types of enzyme activity, such as phagocytosis, clotting, and cell adhesion (the integrins needed for attachment to substrate require Ca++). Because this effect is severe, it is not intended to leave cells in CMF (calcium-magnesium-free) solutions for long.

As Ashfaq stated, for injection you want the cells well dispersed. Once in situ the cations will be readily restored, but if you try to inject sticky clumps, many of the cells will never make it through the needle. Simple test: stain the syringes of CM+ and CMF injections with trypan blue and see what is left: I doubt you will be happy. (Note: naïve syringes, never used before, will be charged to attract cells—coat with a flush of l-lysine first).

It is helpful to read a background text on cell culture to understand the chemistry of the cells you’re manipulating—what they can and can’t (or won’t) do is so dependent on their environment, and understanding the hours it may take for biochemical recovery from a suspension medium may be critical to your results. Be sensitive to that term CMF: many cell culture media labels may say only that to describe the modification.