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Reproductive Immunology Unit
Department of
Obstetrics, Gynecology
& Reproductive Sciences
333 Cedar Street
New Haven CT, 06510
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JAM Assay for Apoptosis
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.
| Reagents |
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| Buffers
and Medias |
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| 1. |
Phosphate
Buffered Saline |
| 2. |
DMEM
media |
| 2. |
RPMI
media |
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| Day
1 |
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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.
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| Day
2 |
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| 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) |
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| Day
3 |
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| 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. |
| 4. |
Analyze
the data using the following formula.
Where: S is spontaneous death and E stands for experimental
death. |
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