Our protocol for using mimosine to arrest human cells in late G1 phase
The basic method, as we use it routinely in the lab, is published here:
Krude, T. (1999). Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner. Exp Cell Res 247, 148-159.
Additional information on using mimosine and other iron chelators to achieve late G1 phase arrest has been published here:
Szüts, D., and Krude, T. (2004). Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage. J Cell Sci 117, 4897-4908.
Further observations and points to consider are the following:
We only use mimosine (purchased from Sigma) for human cells growing attached to the substratum in culture dishes, never for spinner cells. Mimosine treatment results in the formation of dead cells at various percentages, which detach from the substratum. This debris is washed off after the treatment, so that it does not contaminate the preparation of cell nuclei.
We prepare a 10mM stock solution of mimosine in standard culture medium (DMEM/FCS/antibiotics/fungizone) by rotating the suspension for several hours at 37 degrees Celsius, or even overnight at room temperature (i. e. NOT in the cold room). This is essential as mimosine dissolves very slowly at this concentration. Then the solution is sterile-filtered through 0.2µm membranes and can be used directly by adding it to the tissue culture medium at a concentration of 0.5mM. However, we have seen sporadically with a very few batches of mimosine that we need to add up to 0.7mM to achieve a full G1 phase block.
A treatment of proliferating cells for 24h is usually sufficient.
We have obtained a late G1 phase arrest in all human cell lines tested so far, including: HFF foreskin fibroblasts, WI38 embryo lung fibroblasts, SKOV3 ovarian carcinoma, EJ30 bladder carcinoma and HeLa cervical carcinoma cells.