CD95 (Fas)-induced apoptosis of human Jurkat cell line using EOS9.1 mAb

When studying induction of apoptosis via a cell surface molecule, it is important to first ascertain surface expression of the molecule of interest by cell line or cell population in question.  This can be achieved by performing flow cytometry or immunoprecipitation/Western blotting assays.  The other factor important in achieving optimal results is the degree of sensitivity of cells to the antibody-induced apoptosis. 

As an example, CD95 (Fas)-induced apoptosis of human Jurkat cell line by monoclonal antibody EOS9.1 is described here as a general positive control for eBioscience apoptosis detection systems.  The Jurkat cells from different sources used in various laboratories demonstrate large variability in their degree of sensitivity to the Fas-induced apoptosis.  For best results, cells should be in their exponential log phase with viability rate of greater than 95% when they are used for this assay.

What you need:

*Materials:

-Jurkat cells from ATCC (E6-1, cat.no. TIB-152)

-RPMI-1640/10%FCS

-Anti-human CD95, Clone EOS9.1, cat.no. 16-0958

-24-well culture plate (Costar cat.no. 3526)

-12 x 75 mm flow cytometry centrifuge tubes (Falcon cat.no. 2008)

-Apoptosis detection kit (Apo-Direct cat.no. 88-6611 or Apo-BrdU kit cat.no. 88-6671)

*Instruments:

-Centrifuge

-Pipettes and pipettors

-Flow cytometer or a fluorescence microscope

Experiment Duration: 

1/4 hour for cell preparation

The incubation period is variable and should be determined empirically by the researcher (a suggested period to try is 16-18 hours)

Degree of difficulty:

Easy

Method:

1) Prepare a single-cell suspension of the Jurkat cell line.  Place 5 x 10(5) cells per ml of media in a 24-well plate for each sample.  Use 2-3 wells per condition to allow enough numbers of cells for the following staining and analysis.

2) Treat cell suspension with a titration of EOS9.1 mAb (a range of 1 ug/ml to 0.06 ug/ml is recommended.  Cells in wells treated with medium only can be used as unstimulated cells.

3) Incubate overnight (16-18 hours or as long as your special experimental question requires).

4) Pool the cells from each condition and spin cells for 3-4 minutes at 300-400xg.

5) Aspirate the supernatant and follow instructions of your favorite kit for detection of apoptosis of treated cells.