Protocols and Applications Guide (US Letter Size) - Promega

||| 3Apoptosis
VIII. General Protocols for Inducing Apoptosis in Cells
Apoptosis may be induced in experimental systems through
a variety of methods, including:
Treating cells with the protein synthesis inhibitor,
anisomycin, or the DNA topoisomerase I inhibitor,
camptothecin, induces apoptosis in the human
promyelocytic cell line HL-60 (Del Bino et al. 1991; Li et al.
1995; Gorczyca et al. 1993; Darzynkiewicz et al. 1992).
Withdrawal of growth factors induces apoptosis of growth
factor-dependent cell lines. For example, NGF-deprivation
of PC12 cells or sympathetic neurons in culture induces
apoptosis (Batistatou and Greene, 1991).
In vitro treatment with the glucocorticoid, dexamethasone,
induces apoptosis in mouse thymus lymphocytes (Gavrieli
et al. 1992; Cohen and Duke 1984).
Activation of either Fas or TNF-receptors by the respective
ligands or by cross-linking with agonist antibody induces
apoptosis of Fas- or TNF receptor-bearing cells (Tewari and
Dixit 1995).
A. Anti-Fas mAb Induction of Apoptosis in Jurkat Cells
1. Grow Jurkat cells in RPMI-1640 medium containing
10% fetal bovine serum in a humidified, 5% CO2
incubator at 37°C.
2. Suspend the cells in fresh medium at a concentration
of 1 × 105 cells/ml. After two to three days of incubation
in a 37°C, 5% CO2 incubator, harvest the cells by
centrifugation at 300–350 × g for 5 minutes.
3. Resuspend cells in fresh medium to 5 × 105 cells/ml and
add anti-Fas mAb to a final concentration of
0.05–0.1µg/ml. Incubate for 3–6 hours in a 37°C
incubator. As a negative control, incubate untreated
cells (no anti-Fas mAb) under the same conditions.
(Stop here for homogeneous assay, or plate the cells in
a 96-well plate.)
4. Harvest the cells by centrifugation at 300–350 × g for 5
minutes.
5. Remove all medium and resuspend cells in PBS.
6. Repeat centrifugation and resuspend the cell pellet in
PBS to 1.5 × 106 cells/ml.
B. Anisomycin-Induced Apoptosis in HL-60 Cells
Treatment with the protein synthesis inhibitor, anisomycin
induces apoptosis in the human promyelocytic cell line
HL-60.
1. Grow HL-60 cells in RPMI-1640 medium containing
10% fetal bovine serum in a humidified 5% CO2
incubator at 37°C.
2. Adjust the cell density to 5 × 105 cells/ml and treat with
anisomycin at a final concentration of 2µg/ml (dissolved
in DMSO). Incubate for 2 hours in a humidified 5% CO2
Protocols & Applications Guide
www.promega.com
rev. 3/07
incubator at 37°C. Treat negative control cells with an
equal volume of DMSO, and incubate under the same
conditions.
3. Harvest the cells and resuspend in PBS to 1.5 x 106/ml.
C. Staurosporine-Induced Apoptosis in SH-SY5Y Neuroblastoma
Cells
1. Culture cells in a 1:1 mixture of Ham’s F12 nutrients
and minimal essential medium supplemented with 10%
fetal bovine serum (FBS), 100IU/ml penicillin and
100mg/ml streptomycin in an atmosphere of 95% air
and 5% CO2 at 37°C.
2. Allow cells to reach 70% confluence. Trypsinize to
release cells from the flask, and plate in a 96-well plate
in 45% MEM, 45% F12K and 10% FBS.
3. After 24 hours, treat cells with 100µl of 3.125µM
staurosporine in DMSO.
4. Incubate with staurosporine for 24 hours before
performing cell-based assay.
IX. References
Batistatou, A. and Greene, L.A. (1991) Aurintricarboxylic acid
rescues PC12 cells and sympathetic neurons from cell death caused
by nerve growth factor deprivation: Correlation with suppression
of endonuclease activity. J. Cell Biol. 115, 461–71.
Boatright, K.M. et al. (2003) A unified model for apical caspase
activation. Molecular Cell 11, 529–41.
Bossy-Wetzel, E. and Green, D.R. (2000) Detection of apoptosis:
Annexin V labeling. In: Meth. Enzymol. Reed, J.C. ed. 332, 15–18.
Chan, F.K. et al. (2000) A domain in TNF receptors that mediates
ligand-independent receptor assembly and signaling. Science 288,
2351–54.
Cohen, J.J. and Duke, R.C. (1984) Glucocorticoid activation of a
calcium-dependent endonuclease in thymocyte nuclei leads to cell
death. J. Immunol. 132, 38–42.
Cotter, T.G. and Curtin, J.F. (2003) Historical perspectives. In:
Essays in Biochemistry. Cotter, T.G. et al. eds. 39, 1–10.
Csiszar, A. et al. (2004) Proinflammatory phenotype of coronary
arteries promotes endothelial apoptosis in ageing. Physiol. Genomics
17, 21–30.
Daniel, P.T. et al. (2003) Guardians of cell death: The Bcl-2 family
proteins. In: Essays in Biochemistry. Cotter, T.G. et al. eds. 39, 73–88.
Darzynkiewicz, Z. et al. (1992) Features of apoptotic cells measured
by flow cytometry. Cytometry 13, 795–808.
Del Bino, G. et al. (1991) The concentration-dependent diversity
of effects of DNA topoisomerase I and II inhibitors on the cell cycle
of HL-60 cells. Exp. Cell Res. 195, 485–91.
Earnshaw, W. et al. (1999) Mammalian caspasese: Structure,
activation, substrates, and functioins during apoptosis. Annu. Rev.
Biochem. 68, 383–424.
PROTOCOLS & APPLICATIONS GUIDE 3-20