Donor Mice

Forty B10.S IFN- γ^-/- mice were used as the donors. These mice were 8-12 weeks aged at the onset of treatment. Twenty mice were treated with 4mg/l mercuric chloride in drinking water and the rest twenty were only given tap water (controls). All mice were sampled before the onset of the study and after 30 days when they were sacrificed. Sacrificed by cervical dislocation and spleen and kidney were collected. Splenic single cells were prepared from Hg- treated and untreated mice.  Kidney was preserved at -70 ⁰C freezers for later use.  

Tregs cell isolation

Treg cells, CD4⁺CD25⁺   and CD4⁺CD25^- cells were isolated from spleen by using the magnetic cell separating kits by Miltenyi Biotec, Germany. Splenic single cell suspension from Hg treated, and untreated mice were prepared by mechanically disrupting as described by Johansson 1997, silver IA. CD4⁺CD25⁺   and CD4⁺CD25^- cells were purified from splenic single cell suspension using a magnetic cell separator. Purity of Tregs and non- Tregs were examined by flow cytometer analysis. Purified CD4⁺CD25⁺   and CD4⁺CD25^-   cell from both hg treated and untreated mice were sorted in four groups each and transferred to new set of mice (fig.1).  

Recipient Mice

Recipient mice used were B 10.S wild-type mice. Purified CD4⁺CD25⁺   and CD4⁺CD25^-    cells from the donor mice were totally divided into 8 set and injected in the recipient mice.  10x10⁵ cells per mouse in 0.1 ml PBS was injected via intraperitoneal injection. 39 mice were used for the purpose in which 19 was water treated, and 20 were mercury treated. 4mg mercuric chloride in one litre water was used for treatment. Control mice received only tap water. Blood samples were collected from the mice at 0 weeks (just taken before the onset of treatment), 5, 7, 9, 11, 13, and 15 weeks after onset of treatment. The mice were sacrificed by cervical dislocation after 15 weeks and kidney, and spleen was collected and stored at    -70 freezers, which will be preserved for later use.