New protocol in blue!

Each substrate molecule contains a peptide homodoubly labeled with a fluorophore. The cleaved substrate has the following fluorescence peak characteristics:  _ex= 505 nm and _em= 530 nm. (The fluorescence of the precleaved fluorogenic protease substrate is not completely quenched.)

The main peptide amino acid sequence is GDEVDGI.

Before storing: Please centrifuge the reagent-containing vials lightly to remove any liquid from caps, then store at -10 to -20 ^oC.

Upon thawing: Shake until the solution appears homogeneous.

Incubation conditions:

Do not fix cells that have been previously incubated with the substrate for antibody labeling or labeling with other reagents. Remove all steps involving fixation or permeabilization from your protocol.

1. Treat target cells with chosen apoptosis-inducing reagent and/or inhibitor. (See D below.)

2. Aliquot cells into 1.5 to 2.0 ml microcentrifuge tubes, then centrifuge and remove all of the culture medium in order to minimize substrate dilution. Use a gentle vacuum suction equipped with a capillary tip to completely remove the medium from microfuge tubes with the centrifuged cell pellets.

3. To each of the centrifuged cell pellets, add 50 to 75 ul of 10 uM substrate solution (and add 5 ul of FCS, if 10% FCS is appropriate). The cell number in these solutions should be between 0.5 and 1 million per sample (See A and B below). Mix suspension containing cells and substrate by flicking tubes with finger tips. Do not vortex tubes containing cells. Apoptotic cells can be “fragile’”.

4. Incubate tubes at 37⁰ C for 60 minutes before flow cytometric analysis. (See C below.)

5. Avoid direct light to substrate and exposure to extremes of pH. Keep substrate at physiological pH.

6. Recommended flow cytometer setting: FL1 channel of a BD instrument with excitation at 488 nm. Use Fl3 for PI.

7. Wash cells once by adding 1 ml of ice cold flow cytometry dilution buffer, centrifuging, and removing all of buffer. Loosen the cells pellets by flicking the tubes with finger tips and then resuspending the loosen pellets in 1 ml of fresh dilution buffer. Do not vortex tubes containing cells, but rather flick tubes with finger tips to resuspend cells. Apoptotic cells can be “fragile”. (See D.)

8. Keep cell suspension on ice until analysis by flow cytometry.

9. All samples should be analyzed within 60 to 90 minutes after the end of the 37 ^oC incubation.

10. After collecting data by the preceding procedure, one can delete PI(+) cells if a drop of a 5-10 ug/ml propidium iodide (PI) solution is added and samples are rerun on the flow cytometer (final PI concentration of @200ng/ml) (See D). Reanalysis should be within 5 minutes of PI addition. Alternatively, if an additional 2.0 ml of flow cytometry dilution buffer (or PBS) is added to each sample, then cells that would be PI(+) will shift to a lower intensity range due to the leakage of intact PhiPhiLux substrate. Thus, the fluorescence of dead cells, i.e., those that would be PI(+), may show lower fluorescence intensity in the FL1 channel than will apoptotic PI(-) or non-apoptotic cells. (See D below.)

A. The cell density during incubation with the substrate can be as high as 0.5 to 1 million cells per sample. It is recommended that a control sample with the cells taken directly from a culture with cells in log phase be included in the assay to test whether there is any high cell density-induced apoptosis.

B. In certain settings, one may be able to use the substrate at a concentration lower than 9 uM [addition of FCS to a final concentration of 10% v/v would lower the substrate concentration to 9 uM.]. However, the recommended final substrate concentration should not be less than 9 uM.

C. Viable cell uptake of the substrate reaches a near maximum by 15 to 20 minutes at 37 ^oC. as stated above, the incubation time may vary with cell type.

D. We recommend that in order to identify PI(-) apoptotic and uninduced cell populations, one analyze samples, first, without PI addition and then the same samples after PI addition. When one compares the dot plots of FL1 vs FL3 (or FL2) of these two samples, one can easily identify the PI(-) cell population as those cells that remain in the same location in the FL1 vs FL3 [FL2] dot plot after PI has been added. Place a gate around the PI(-) population and plot this gated FL1 histogram. This histogram should show both uninduced and apoptotic cells. One should avoid use of a threshold-based (orthogonal) gate for PI(+) cells, i.e., designation of cells above a certain FL2 or FL3 channel number as PI(+), since some very bright PI(-) apoptotic cells may appear in a region defined as PI(+). Be sure that the sample dilution volume is the same for all samples.

If one observes the appearance of a very low fluorescence intensity population, i.e., lower than the uninduced cell population, then more than likely the samples have been overtreated with inducing agent or conditions that over induce the sample. In order to see the brighter apoptotic cell populations in histograms, these apoptotic cells must retain their membrane integrity. Pleae note that caspase-cleaved substrate fragments which generate positive signal in the PI(-) apoptotic cells are retained since the fragments diffuse through intact membranes significantly slower than the intact substrate. This differential diffusion rate will be lost when the cells loose their membrane integrity. When membrane integrity is lost, the cleaved substrate fragments leak out of such cells and these cells appear dark.

Generally, cells in samples with a high percentage of PI(+) or TUNEL assay positive cells will be in late apoptosis and/or necrosis. Use of PhiPhiLux is optimal with samples containing a low percentage of PI(+) cells. In most cases we recommend lowering the inducing agent concentration rather than simply looking for an earlier time point. Appropriate time points for PhiPhiLux analysis of apoptotic cells should be such that a large percentage of viable PI(-) cells is present and the samples contain both uninduced cells plus caspase(+)/PI(-) cells. Examination of cells under a fluorescence microscope may assist in determining exact apoptosis inducing conditions.

Updated 09/20/00

Incubation conditions:

Do not fix cells that have been previously incubated with the substrate for antibody labeling or labeling with other reagents. Remove all steps involving fixation or permeabilization.

1. Treat target cells with chosen apoptosis-inducing reagent and/or inhibitor. (See D of Application #1: Flow Cytometry Application.)

2. (a) For suspension cells, aliquot into 1.5 to 2.0 ml microcentrifuge tubes, then centrifuge and remove as much the culture medium as possible in order to minimize the substrate dilution. (b) For adherent cells, remove all culture medium.

3. (a) For suspension cells, to each of the centrifuged cell pellets, add 50 to 75 ul of 10 uM substrate solution (and add 5 ul of FCS, if 10% FCS is appropriate). The cell number in these solutions should be between 0.5 and 1 million per sample (See A and B in Application #1: Flow Cytometry Application). Mix suspension containing cells and substrate by flicking tubes with finger tips. Do not vortex tubes containing cells. Apoptotic cells are “fragile”. (b) For adherent cells, add enough substrate solution to completely cover the monolayer or individual adherent cells. With either configuration be sure to remove all medium before addition of substrate-containing solution to minimize dilution of substrate.

4. Incubate suspension of cell samples in tubes or adherent cells at 37 ^oC for 30 to 60 minutes. (For adherent cells we recommend a 35 mm cell culture dish with a glass coverslip attached to the bottom side). Exact incubation times are cell type and inducer specific.

5. Avoid direct light to substrate and exposure to extremes of pH. Keep substrate at physiological pH.

6. Immediately following incubation with PhiPhiLux: (a) for suspension cells dilute with 1.5 ml of the physiological buffer, e.g., PBS at 4 ^oC, centrifuge, and replace the supernatant with 1.5 ml of fresh buffer (at 4 ^oC). Repeat this cell washing process two to four more times depending on your fluorescence microscope’s lamp power and detection capability. Check cells after each wash under fluorescence microscope to see if the background fluorescence is dark enough to distinguish between untreated control and treated cells. (b) for adherent cells remove PhiPhiLux-containing medium and wash gently with buffer. Perform a similar number of cell washing cycles as suspension cell samples. After each cycle make observation under fluorescence microscope to evaluate the background fluorescence level. Take special care in washing adherent cells since apoptotic cells are generally more easily detached from the plate than nonapoptotic cells.

7. Recommended microscopy settings are fluorescein filters.

Please contact us for ordering information for sterile 35 mm culture dishes with glass centers for use with inverted fluorescence microscopes.

References (updated 05/31/00):

On Exciton theory used in fluorogenic protease substrate design:

1. B.Z. Packard, D.D. Toptygin, A. Komoriya, and L. Brand Profluorescenct protease substrates: intramolcular dimers described by the exciton model. Proc. Natl. Acad. Sci. (USA) 93: 11640-11645 (1996).

2. B.Z. Packard, D.D. Toptygin, A. Komoriya, and L. Brand. The design of fluorogenic protease substrates guided by exciton theory. Meth. Enzym. 278: 15-28 (1997).

3. B.Z.Packard, A. Komoriya, D.D.Toptygin, and L. Brand. Structural characteristics of fluorophores which form intramolecular H-type dimers in a protease substrate. J. Phys. Chem. B 101: 5070-5074 (1997).

4. B.Z. Packard, D.D. Toptygin, A. Komoriya, and L. Brand. Characterization of fluorescence quenching in bifluorophoric protease substrates. Biophys. Chem. 67: 167-176 (1997)

5. B. Z. Packard, D.D. Toptygin, A. Komoriya, and L. Brand. Intramolecular resonance dipole-dipole interactions in a protease substrate. J. Phys. Chem. B 102: 752-758 (1998).

6. B.Z. Packard, A. Komoriya, V. Nanda, and L. Brand. Intramolecular excitonic dimers in protease substrates: modification of the backbone moiety to probe the H-dimer structure. J. Phys. Chem. B 102: 1820-1827 (1998).

7. A. Komoriya, B. Z. Packard, M. J. Brown, M.-L. Wu, and P. A. Henkart. Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-permeable Fluorogenic Caspase Substrates. J. Exp. Med. 191:1819-1828 (2000)

On Caspase-3 nomenclature:

8. E.S. Alnemri et al. Human ICE/CED-3 Protease Nomenclature. Cell 87: 171 (1996)

On DEVD-based substrate and inhibitor characteristics:

9. A. Sarin, M.-L. Wu, and P.A. Henkart. Different Interleukin-1b Converting Enzyme (ICE) family protease requirements for the apoptotic death of T-lymphocytes triggered by diverse stimuli. J. Exp. Med. 184: 2445-2450 (1996)

10. D.W. Nicholson et. al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43 (1995).

11. N.A. Thornberry, T.A. Rano, S.Roy, J.P. Vaillancourt, K.T. Chapman, and D.W. Nicholson. A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B Functional Relationships established for Key Mediators of Apoptosis. J. Biol. Chem. 272:17907-17911 (1997)

Intracellular Caspase-3-like activities determination using PhiPhiLux:

12. H. Hirata, A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okasaki, K. Yamamoto, and M. Sasada. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187: 587-600 (1998)

13. J. M. Zapata, R. Takahashi, G.S.Salvesen and J.C. Reed. Granzyme release and caspase activation in activated human T-lymphocytes. J. Biol. Chem. 273: 6916-6920 (1998).

14. R.M. Siegel, D.A. Martin, L.Zheng, S.Y. Ng, J. Cohen, and M.J. Lenardo. Death-effector filaments: novel cytoplasmic structures that recruite caspases and trigger apoptosis. J. Cell Biol. 141: 1243-1253 (1998).

15. L. Guedez, W.G. Stetler-Stevenson, L. Wolff, J. Wang, P. Fukushima, A. Mansoor, M. Stetler-Stevenson. In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1. J. Clin. Invest. 102: 2002-10 (1998).

16. P.G. Ekert, J. Silke and D.L. Vaux. Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors. EMBO J. 18: 330-338 (1999).

17. D.M. Finucane, E. Bossy-Wetzel, N.J. Waterhouse, T.G. Cotter and D.R. Green. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274: 2225-2233 (1999).

18. G.I. Perez, X-J. Tao and J.L. Tilly. Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. Molecular Human Reproduction 5: 414-420 (1999).

19. R. Robles, X-J. Tao, A.M. Trbovich, D.V. Maravei, R. Nahum, G.L. Perez, K.L. Tilly and J.L. Tilly. Localization, regulation and possible consequence of apoptotic protease activating factor-1(Apaf-1) expression in granulosa cells of the mouse ovary. Endocrinology 140: 2641-2644 (1999).

20. H. Kanuka, S. Hisahara, K. Sawamoto, S. Shoji, H. Okano and M. Miura. Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: Implicated mechanisms for caspase activation. Proc. Natl. Acad. Sci. 96: 145-150 (1999).

21. B. Huppertz, H.-G. Frank, and P. Kaufmann. The apoptosis cascade -- morphological and immunohistochemical methods for its visualization. Anat. Embryol. 200: 1-18 (1999).

22. B. Huppertz, H.-G. Frank, F. Reister, J. Kingdom, H. Korr, and P. Kaufmann. Apoptosis Cascade Progressess during Turnover of Human Trophoblast: Analysis of Villous Cytotrophoblast and Syncytial Fragments in Vitro. Laboratory Investigation 79:1687-1702 (1999).

23. K.J. Harvey, D. Lukovic, and D.S. Ucker. Caspase-dependent Cdk activity is a requisite effector of apoptotic death events. J. Cell Biol. 148:59-72 (2000).

24. A. Komoriya, B.Z. Packard, M.J. Brown, M.-L. Wu, and P.A. Henkart. Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-permeable Fluorogenic Caspase Substrates. J. Exp. Med. 191:1819-1828 (2000).

Updated 9/20/00. Copyright © 1998-2001 OncoImmunin Inc.  All rights reserved.