Materials and Reagents

Mice
Clodronate (dichloromethylene diphosphonic acid)-loaded liposomes (VUmc)
PBS-containing liposomes (VUmc)
Phosphatidyl choline (Lipoid GmbH, catalog number: D-67065)
Cholesterol (Sigma-Aldrich, catalog number: C8667 )
Liposomes (see Recipes)
Liposomes suspended in sterile phosphate buffered saline (see Recipes)

Equipment

Syringe (10 ml and 1 ml) (BD, catalog numbers: 309604 and 305111 )
26 G 1/2 needles (BD, catalog number: 309659 )

Procedure

Twenty, 8-week-old mice were randomly divided into two groups.
One group of mice were intravenously injected t using 1-ml syringe with 26 G 1/2 needles, twice per week with clodronate-loaded liposomes in 150 µl for the first month and 100 µl following the first month’s injection to reduce possible side effects, such as infection, live toxicity et al. The mice were treated for 3 months totally.
The second group was treated with PBS-liposomes.
At different time points (such as 2, 4, 8 weeks after treatments) of the experiment, one or two mice from each group will be euthanized to examine inflammation and other interesting analysis. Based on our experiments, the FACS analysis detected around 90% of macrophage reduction in the group that treated with the clodronate-loaded liposomes versus the control group after 2 weeks treatment, and this reduction kept to the end of the experiments.
During the whole treatment period, the treated mice need to be closely monitored for the possible signs and symptoms such as weight loss, yellow, bloody, or tar-colored stool, dark urine color, diarrhea, fatigue etc.
At the end of the experiment, (depend on the experiment), the mice were euthanized and the lung tissues were collected for various analyses.

Recipes

Notes:

Based on the instructions from company.
The clodronate-loaded liposomes and PBS-containing liposomes are ready for use, but the solutions that usually kept in 4 °C need to be warmed up to room temperature before injections.

Liposomes
Liposomes are artificial lipid spherules made of:
1. Phosphatidyl choline
2. Cholesterol
Phosphate buffered saline (PBS)
1. 10 mM Na₂HPO₄
2. 10 mM NaH₂PO₄
3. 140 mM NaCI

Acknowledgments

We thank Yinling Hu, Robert H. Wiltrout and Jami Willette-Brown from Frederick National Laboratory for Cancer Research for their support and suggestions on the experimental design. This work was supported by the National Cancer Institute (ZIA BC 011212 and ZIA BC 011391 to Y.H.).

References

Xiao, Z., Jiang, Q., Willette-Brown, J., Xi, S., Zhu, F., Burkett, S., Back, T., Song, N. Y., Datla, M., Sun, Z., Goldszmid, R., Lin, F., Cohoon, T., Pike, K., Wu, X., Schrump, D. S., Wong, K. K., Young, H. A., Trinchieri, G., Wiltrout, R. H. and Hu, Y. (2013). The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. Cancer Cell 23(4): 527-540.