The Jam assay for quantification of the induction of apoptosis is a novel test developed by Matzinger in 1991 18. It is based on the principle that dying cells often degrade their DNA into small fragments, which are not retained by the filter paper during a typical [3H] Thymidine proliferation assay. The Jam assay measures the DNA retained by living cells rather than the cellular component lost by dying cells. 8,13.
|Buffers and Medias|
|1.||Phosphate Buffered Saline|
Plan Experiment, select and grow effector and target cells. In our system we used as effector cell the choriocarcinoma Jar cell line that express high levels of FasL. As target cells we use the T cell line Jurkat cells which express Fas receptor on its surface. In a 96 well plate; add 200,000 Jar cells in the firs well in 200 µl of normal DMEM media. Prepare serial dilutions of the cells, either 2:1 or 4:1. Incubate overnight.
|1.||Prepare the target Jurkat cells. Make sure you separate them in two groups: Labeled and non-labeled. Label the cells with [3H] Thymidine: 5uCi/ml and 400.000 Jurkat cells. Incubate the cells at 37°C for 6 h.|
|2.||Wash the labeled Jurkat cells 3 times with RPMI media, in1.5 ependorff tubes (to prevent loss of cells)|
|3.||Mark the plate into S, T and E (T: total incorporation). Add the Jurkat cells to the wells containing the attached Jar cells and incubate for 24h. (Use triplicates)|
|1.||Harvest the cells into glass paper filters. Then put them in scintillation tubes. Follow the instructions given for the specific harvest machine.|
|2.||Make sure the cells in the group labeled as "total" are lysed by freezing and thawing with liquid nitrogen. Put the content in scintillation tubes.|
|3.||Collect the filters into scintillation tubes and read them in a beta counter.|
Analyze the data using the following formula.
Where: S is spontaneous death and E stands for experimental death.