Presently, I am focusing on an experimental metastasis model that utilizes 4T1 breast cancer cells.
We typically trypsinize the cells from the dish and then pipette them to form a single cell solution. We subsequently count the cells and dilute them to our desired concentration, ranging from 100k/ml to 1000k/ml. The cells are then aliquoted into an insulin syringe (100 ul), placed on ice, and transferred to the animal facility for tail vein injection. Subsequently, we have been imaging metastatic tumor formation using bioluminescence imaging, specifically with the Bruker Xtreme II in vivo imager. While we have indeed been able to observe lung tumor formation via this in vivo imaging technique, a few challenges have arisen, and I was hoping you could provide some insight or guidance.
Firstly, our experiments have shown multiple occurrences of tail tumors. Some cases present as exclusive tail tumors, while others coexist with lung metastatic tumors. I am curious if you have observed a similar incidence rate of tail tumors following tail vein injections? If so, would you have any recommendations to potentially decrease the occurrence rate of these tail tumors? Do you recommend a filtration step with a cell strainer before the cell counting? Also, would it be beneficial to leave a small amount of EDTA in the injection solution (PBS) to prevent the formation of cell clumps?
Secondly, we've encountered issues where some lung metastatic tumors are not visible during in vivo imaging, yet are detectable post-dissection. Follow-up in vitro imaging has confirmed these tumors are indeed capable of emitting a bioluminescence signal. This leads me to assume that the issue lies in either the strength of the bioluminescence signal or the tumor's size or deep placement within the lung. If you have experienced a similar situation, I would be grateful for any advice or suggestions to address this issue. About the cells we are using, we have generated our own BLI-4T1 cells and have concentrated the higher expression cells with FACS. We always use fresh stock from the liquid nitrogen, and these cells have tested negative for mycoplasma. To reduce light diffraction, we shave the chest hair of our mice. However, we have considered obtaining commercially available cells. Are there any particular sources you would recommend?
I look forward to hearing your thoughts on these matters. Thank you in advance for your advice.
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