A-2014A

AAPPLICATION PPLICATION IINFORMATION NFORMATION Flow Cytometry

EVALUATION OF APOPTOSIS WITH ANNEXIN V AND PROPIDIUM IODIDE OR 7-AMINO-ACTINOMYCIN D Jay Enten, MaryLyn Monson Cellular Analysis Business Center, Beckman Coulter, Inc., Miami, FL 33196

Introduction

Procedure

Changes in cellular morphology including cell shrinkage, nuclear condensation, and pyknosis, along with biochemical events leading to loss of mitochondrial membrane potential, loss of plasma membrane asymmetry, and DNA cleavage between nucleosomes characterize Apoptosis. Not all cells in apoptosis, however, appear to cleave their DNA strands, and for those that do, the occurrence is late in the apoptotic pathway. The appearance of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell is another parameter, which can be used to detect and measure apoptosis. The presence of PS on the cell surface creates one of the specific signals for recognition and removal of apoptotic cells by macrophages. These PS changes can be detected with the anticoagulant, Annexin V, which has a high affinity for binding to PS. As the apoptotic process progresses, cell membrane integrity is lost. Using DNA specific viability dyes, such as Propidium Iodide (PI) or 7-AminoActinomycin D (7-AAD), and the Cell Lab Quanta™ it is possible to distinguish between early apoptotic, late apoptotic, and dead cells. The Cell Lab Quanta is a flow cytometry system designed to simultaneously measure Electronic Cell Volume (EV) and fluorescence. In this application note, a simple and rapid protocol is described that provides a powerful tool for the study of apoptosis.

1. Wash cell suspensions with ice cold medium or PBS and centrifuge at 4ºC and 500 x g for 5 minutes. Discard the supernatant. Resuspend the cell pellet to 5 x 106–10 x 106 cells/mL in ice-cold 1X Binding Buffer. Keep on ice. 2. Use 100 µL of the cell suspension to prepare the following controls and samples:

Materials and Methods • Flow Check Fluorospheres™ PN 6605359 • Annexin V-FITC/7-AAD Kit PN IM3614 • Annexin V-FITC/PI Kit PN IM2375

Untreated TUBE 1 2 3 4 5

Cells Only X

7-AAD or PI

Treated

Annexin V-FITC

7-AAD or PI

Annexin V-FITC

X X X

X X

X

3. Follow the package insert directions of the kit being used for antibody and dye reagent volumes and incubation times. 4. Add 400 µL 1X binding buffer. Transfer to sample cups and analyze within 30 minutes on the Cell Lab Quanta. Perform instrument alignment with Flow Check Fluorospheres.

Apoptosis Protocol Use tube 1 to establish proper instrument settings: For volume, bring the entire cell population on scale (Figure 2). For fluorescence, locate FL1 and FL2 populations completely on scale and close to the edge of the first log decade. With the settings from tube 1, use tubes 2 and 3 to establish proper compensation (Figures 3, 4). Applying the settings attained from tubes 1, 2, and 3, utilize tube 4 to establish proper placement of a quadrant region (Figure 5). Record tube 5 results for each quadrant: Quadrant 1–Dead Cells, Quadrant 2–Late Apoptotic Cells, Quadrant 3–Viable Cells, Quadrant 4– Early Apoptotic Cells (Figure 6).

Instrument Configuration

9. Verhoven, B., Schlegel, R. A., Williamson, P.: 1995. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182: 1597–1601. 10. Vermes, I., Haanen, C., Reutelingsperger, C. P. M.: 1995. A novel assay for apoptosis: Flow Cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods 180: 39–52. 11. Castedo, M., Hirsch, T., Susin, S. A., Zamzami, N., Marchetti, P., Macho, A., Kroemer, G.: 1996. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J Immunol 157: 512–521. 12. Aubry, J. P., Blaecke, A., Lecoanet-Henchoz, S., Heannin, P., Herbault, N., Caron, G., Moine, V., Bonnefor, J. Y.: 1999. Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry 37: 197–204. 13. Koopman, G., Reutelingsperger, C. P. M., Kuijen, G. A. M., Keehnen, R. M. J., Pais, S. T., van Oers, M. H. J.: 1994. Annexin V for flow Cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 5, 84: 1415–1420. 14. Van Engeland, M., Ramaekers, F. C. S., Schutte, B., Reutelingsperger, C. P. M.: 1996. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24: 131–139. 15. Pepper, C., Thomas, A., Tucker, H., Hoy, T., Bentley, P: 1998. Flow Cytometric assessment of three different methods for the measurement of in vitro apoptosis. Leukemia Res 22: 439-444. 16. Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Bubertret, L., Sigaux, F., Zhang, Y., Sasportes, M.: 1997. Acquisition of granzyme B and Fas Ligand Proteins by human keratinocytes contributes to epidermal cell defense. J Immunol 159: 5293-5300. 17. van den Eijnde, S., Boshart, L., Reutelingsperger, C. P. M., De Zeeuw, C. I., Vermeij-Keers, C.: 1997. Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis. Cell Death Differentiation 4: 311–316. 18. van den Eijnde, S. M., Boshart, L., Baehrecke, E. H., De Zeeuw, C. I., Reutelingsperger, C. P. M., Vermeij-Keers, C.: 1996. Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved. Apoptosis 3: 9–16.

• 488 nm Laser • 560 Short Pass Dichroic • 525/40 Band Pass (FL1) Annexin V-FITC • 570 Long Pass (FL2) PI or 7-AAD

Selected References 1. Kerr, J. F .R., Wyllie, A. H., Currie, A. R.: 1972. Apoptosis, a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26: 239–245. 2. Wyllie, A. H., Morris, R. G., Smith, A. L., Dunlop, D.: 1984. Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142: 62–77. 3. Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakerling, A. E., Walker, P. R., Sikorska, M.: 1993. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 9, 12: 3679–3684. 4. Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J., van Schie, R. C. A. A., LaFace, D. M., Green, D. R.: 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus. Inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182: 1545–1557. 5. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., Henson, P. M.: 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 7, 148: 2207–2216. 6. Diaz, C., Schroit, A. J. : 1996. Role of translocases in the generation of phosphatidylserine asymmetry. J Membrane Biol, 151: 1–9. 7. Homburg, C. H. E., de Haas, M., von dem Borne, E. G. Kr., Verhoeven, A. J., Reutelingsperger, C. P. M., Roos, D.: 1995. Human neutrophils lose their surface FC γ RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 2, 85: 532–540. 8. De Rosa, L.: 1993. Bone marrow autograft with immunomagnetic beads purging in poor prognosis B lymphoid malignancies. Haematologica 1, 78: 203.

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VIABILITY APPLICATION 7-AAD or PI

Laser 488 nm

Z488/543 RPC (DICHROIC)

460 RBSP

7-AAD OR PI

560 SP (SPLITTER)

PMT FL2

Blocker: 570 LP

PMT FL1

Figure 1. Cell Lab Quanta™ optical configuration with 488 nm diode laser.

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Blocker: 525 BP

104

50

103

PI

25

102

101

100

0 0

200

400

600

800

1000

100

EV

101

102

103

104

Annexin V-FITC

Figure 2. Apoptosis - Annexin V-FITC with Propidium Iodide. Tube 1: Normal Jurkat Cells (Untreated) were used to set Volume Region and non-fluorescent signals from FL1 and FL2 were placed in the first log decade. 104

100

103

PI

50

102

101

100 0

10

0

10

1

10

2

10

3

4

100

10

Annexin V

101

102

103

104

Annexin V-FITC

Figure 3. Apoptosis - Annexin V-FITC with Propidium Iodide. Tube 2: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained only with Annexin V-FITC. 104

50

103

PI

25

102

101

100

0 0

10

1

10

2

10

3

10

100

4

10

PI

101

102

103

Annexin V-FITC

Figure 4. Apoptosis - Annexin V-FITC with Propidium Iodide. Tube 3: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained only with PI.

4

104

104

103 Dead Cells (%)

PI

Viable Late Apoptosis Cells (%) (%)

Early Apoptosis (%)

102 0.3

1.6

84.8

13.3

101

100 100

101

102

103

104

Annexin V-FITC Figure 5. Apoptosis - Annexin V-FITC with Propidium Iodide. Tube 4: Normal Jurkat Cells (Untreated), stained with Annexin V-FITC and PI. 104

103 Dead Cells (%)

PI

Viable Late Apoptosis Cells (%) (%)

Early Apoptosis (%)

102 1.8

26.7

31.2

40.3

101

100 100

101

102

103

104

Annexin V-FITC Figure 6. Apoptosis - Annexin V-FITC with Propidium Iodide. Tube 5: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained with Annexin V-FITC and PI.

5

104

50

103

7-AAD

25

102

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0 0

200

400

600

800

1000

100

EV

101

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103

104

Annexin V-FITC

Figure 7. Apoptosis - Annexin V-FITC with 7-AAD. Tube 1: Normal Jurkat Cells (Untreated) were used to set Volume Region and non-fluorescent signals for FL1 and FL2 in the first log decade. 104

100

103

7-AAD

50

102

101

100

0 10

0

10

1

10

2

3

10

100

4

10

7-AAD

101

102

103

104

Annexin V-FITC

Figure 8. Apoptosis - Annexin V-FITC with 7-AAD. Tube 2: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained only with Annexin V-FITC. 104

100

103

7-AAD

50

102

101

100

0 100

101

102

103

100

104

7-AAD

101

102

103

104

Annexin V-FITC

Figure 9. Apoptosis - Annexin V-FITC with 7-AAD. Tube 3: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained only with 7-AAD.

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104

103

7-AAD

Dead Cells (%)

102

0.6

Viable Late Apoptosis Cells (%) (%)

2.4

85.2

Early Apoptosis (%)

11.7

101

100 100

101

102

103

104

Annexin V-FITC Figure 10. Apoptosis - Annexin V-FITC with 7-AAD. Tube 4: Normal Jurkat Cells (Untreated), stained with Annexin V-FITC and 7-AAD. 104

103

7-AAD

Dead Cells (%)

102

1.4

Viable Late Apoptosis Cells (%) (%)

37.9

22.4

Early Apoptosis (%)

38.3

101

100 100

101

102

103

104

Annexin V-FITC Figure 11. Apoptosis - Annexin V-FITC with 7-AAD. Tube 5: Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated), stained with Annexin V-FITC and 7-AAD.

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