Introduction

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When studying induction of apoptosis via a cell surface molecule, it is important to first ascertain surface expression of the molecule of interest. This can be achieved by performing flow cytometry or immunoprecipitation/Western blotting assays. Another 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. 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.

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

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Materials

• Jurkat cells (clone E6-1, ATCC Cat. No. TIB-152)
• RPMI-1640/10%FCS
• Anti-human CD95, clone EOS9.1 (eBioscience Cat. No. 16-0958)
• 24-well culture plate (Costar Cat. No. 3526)
• 12x75 mm flow cytometry centrifuge tubes (Falcon Cat. No. 2008)
• Apoptosis detection kit (eBioscience Apo-Direct Cat. No. 88-6611 or Apo-BrdU kit Cat. No. 88-6671)

Instruments

• Centrifuge
• Pipettes and pipettors
• Flow cytometer

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)

Method

1. Prepare a single-cell suspension of the Jurkat cell line. Place 5x10⁵ cells per ml of media in a 24-well plate for each sample. Use 2-3 wells per condition to allow enough cells for subsequent staining and analysis.
2. Treat cell suspension with a titration of EOS9.1 mAb (a range of 1µg/ml to 0.06µg/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 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 the Apo-Direct Apoptosis Detection Kit .