Many cytometric methods have been used in recent years to identify cells in different stages of apoptosis (early, middle, and late) (4,7). We describe a new, easy method which allows the simultaneous identification of apoptotic sub-populations and the presence/absence of surface antigens on their plasmatic membrane during apoptotic development. In this protocol we use fluorescein diacetate (FDA) (10) or Annexin V-Fitc (8) to discriminate apoptotic cells and Trypan Blue to identify necrotic cells. Trypan Blue, normally used for the optical detection of dead cells, is also a fluorescent dye emitting a red fluorescence (l> 620 nm) when excited by the blue light of an argon laser tuned on 488 nm wavelength (11) .

2.1 Materials

Human myeloid cell line HL-60
RPMI 1640 (A1)
Phosphate buffer (PBS) pH 7.4 (A1)
Hepes buffer, pH 7.4 (A1)
Fluorescein diacetate (FDA) (A1,A2)
Trypan Blue solution (0.4% v/v) (A1,A2)
Camptothecin (A2)
Annexin V-FITC (A2)
Anti-CD11a PE (A2)
Centrifuge (A3)
Flow cytometer (argon ion laser) (A3)

Note: Below, we describe two protocols in which we utilize HL-60 human tumoral cells. These protocols are also adaptable to different cellular lines (Molt-4, Raji, Jurkat, Daudi and K562), human lymphocytes and different apoptotic triggers (UVB, Staurosporine, hyperthermia, etc.).
 

 1. Induce apoptosis in HL-60 cells in logarithmic growth phase. Incubate cells (1 x 10⁵ cells/ml) with 0.15 µM camptothecin, a topoisomerase I inhibitor, for 6 hours at 37°C in a CO₂ incubator.

 
2. Collect cells in 15 ml centrifuge tubes and wash twice with PBS. Spin at 1500 rpm (300 x g) for 5 minutes.

3. Resuspend cells at 1 x 10⁶ cells/ml concentration in PBS.

4. Incubate 2.5 x 10⁵ cells (250 µl) with 5 µl anti-CD11a antibody labelled with PE for 20 minutes at room temperature.

5. Wash twice with PBS and check the positivity of the control population and apoptotic population for LFA-1 antigen.

6. Add 5 µl of 1µg/ml FDA solution prepared in PBS shortly before use, to cells stained with anti-CD11a PE.

7. Gently resuspend cells and incubate at room temperature for 5 minutes.

8. Add 25 µl Trypan Blue at a final concentration of 800 µM. Incubate for 5 minutes and then quantify the apoptotic and dead cells by flow cytometry.
 

1. Follow steps 1 to 5 of the Basic Protocol replacing PBS buffer with Hepes buffer in every step.
 

2. Add 10 µl of Annexin V- Fitc at working dilution (1 µg/µl) to the stained cells.
 

3. Incubate for 15 minutes on the bench.
 

4. Analyze by flow cytometry and compensate the Annexin V green fluorescence with the anti-CD11a orange fluorescence.
 

5. Add 25 µl Trypan Blue at a final concentration of 800 µM. Incubate for 5 minutes and then quantify the apoptotic and dead cells by flow cytometry.
 

Decreases in some cell surface antigens have been seen in apoptotic cells. This occurs in human lymphocytes with the CD45 antigen (1) in neutrophils for different antigens (5,6,8) and in thymocytes for the CD4 and CD8 antigens (2,13). The reason behind the shedding of antigens during apoptosis is still not clear and could perhaps be correlated with a change in the plasmatic membrane structure. Nevertheless it is possible to postulate a role for this surface antigen in some apoptotic processes or in their inhibition (3,15). Many attempts have been made to find a protocol capable of discriminating the apoptotic stage and the phenotype in homogeneous and heterogeneous cellular populations using dual laser cytometer (2,12,14) or fixed cells (9). Instead, our method works using a single laser cytometer, easy to use and to find in the majority of clinical and research cytometry facilities. Moreover, we always use unfixed cells thus avoiding all problems related to antigen loss or accessibility after the fixation process. Another advantage of using unfixed cells is the possibility to follow the development of apoptosis also using other parameters (e.g. scatter signals) (4) and to check, step by step, the presence of certain antigens on apoptotic subpopulations during the whole apoptotic process. Finally, it is also possible to sort the apoptotic cells and analyze their morphology using microscopy (optical or electronic).
 

This method should not be used if you need fetal calf serum proteins or albumin in your medium. Trypan Blue has a great affinity for proteins and the discrimination between live and dead cells becomes less evident if a large amount of extracellular proteins is present in the assay microenvironment (11). It also important to avoid the exposure of cells to Trypan Blue for a period longer than 30 minutes. In this case it is possible to observe an increase in the dead cell population (Trypan Blue positive) due to the Trypan toxicity.

The FDA staining can sometimes result in a weak fluorescence. This can be due either to an error in the preparation of the FDA staining solution, which should be prepared shortly before use every time the experiment is carried out, or to an insufficient quantity of the non-specific esterase substrate. For this reason, we suggest performing preliminary experiments in order to find the most efficient FDA concentration for the cellular model used.

It is essential to carry out the staining protocol with Annexin V in Hepes buffer since the binding of this protein to the phosphatidylserine is calcium dependent, otherwise it is possible to observe no or a weak increase in Annexin V binding. For this reason Trypan Blue in the alternative protocol is also diluted in Hepes.

Figure 1 shows the analysis of a sample of HL-60 control cells (left panel) and of cells treated with 0.15 µM camptothecin for 6 hours (right panel). In the control sample we observe a population of Trypan Blue positive and FDA negative cells (Nec, 6%) and a population of apoptotic cells with weak green fluorescence (2nd-3rd log decade) and negative for Trypan Blue (Apo, 6%). Both of these populations are normally found in culture. The live cells however, show a strong green fluorescence (4th log decade) (L; 87%). When the cells are exposed to an apoptotic trigger, it is possible, whilst maintaining the same analysis quadrants and the same voltages of the photomultipliers, to observe the modifications which occur in the various subpopulations, previously identified (L, Nec, Apo). In fact, we observe the increase of a few percent in the necrotic population, (Nec, 8%) whilst the FDA dim population, decreasing its fluorescence gradually, increases six-fold (Apo, 36%) and simultantaneously the numeric value of the live population decreases (L, 56%). In Figure 2, the expression of LFA-1 antigen is analyzed in the same population tested for FDA, using a CD11a antibody conjugated with phycoeritrine. In the panel A figure, the expression of the antigen in the control population is shown (lighter line). It is possible to note that almost 95% of control cells is LFA-1 positive (negative cells are dead or apoptotic, data not shown). In the same hystogram we also show the expression of this antigen in the camptothecin-treated HL-60 cells after 6 hours (darker line). In these cells, a strong decrease in the antigen expression occurs. In panel B the antigen expression in the whole camptothecin treated HL-60 cells is shown (lighter line) together with the expression observed only in the apoptotic population (dim FDA, R1 = lower left quadrant, Fig. 1). It is evident that apoptotic cells lost this antigen during apoptotis. If we use Annexin V binding to identify the apoptotic population, we obtain a similar result by drawing a gate around the Annexin V positive - Trypan Blue negative population (Figure 3).


Figure 1
 


Figure 2


Figure 3

 Both protocols, basic and alternative, are very quick to carry out. In both, the cell preparation takes about 20 minutes, antibody binding takes another 30 minutes. The FDA staining needs 5-10 minutes while the Annexin V binding takes 15 minutes. The Trypan Blue staining takes just few seconds but we suggest a time of 5 minutes to stabilize the number of stained cells (dead cells). The total assay time (between 65 and 70 minutes) should be taken into account when time course experiments are performed.

1. Carbonari M., M. Cibiati, M. Cherchi, D. Sbarigia, A.M. Pesce, L. dell’Anna, A. Modica and M. Fiorilli. 1994. Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficency virus infection and cancer chemotherapy by a novel flow immunocytometric method. Blood 83:1268-1277. Chrest F.J., M.A. Buchholz,
2. Y.H. Kim, T.K. Known, and A.A. Nordin. 1993. Identification and quantification of apoptotic cells following anti-CD3 activation of murine G₀ T cells. Cytometry 14:883-890. Curnow S.J., M. Barad, M. N. Brun-Roubereau and A.M.
3. Schmitt-Verhulst. 1994. Flow-cytometric analysis of apoptotic and non-apoptotic T-cell receptor-transgenic thymocytes following in vitro presentation of antigen. Cytometry 16:41-48.
4. Darzynkiewicz Z., G. Juan, X. Li, W. Giorczyca, T. Murakami, and F. Traganos. 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20.
5. Dransfield I., A.M. Buckle, C. Haslett, and N. Hogg. 1994. Neutrophil apoptosis is associated with a reduction in CD16 (FcgRIII) expression. J.Immunol.153:1254-1263.
6. Dransfield I., S.C. Stock and C. Haslett. 1995. Regulation of cell adhesion molecules and function associated with neutrophil apoptosis. Blood 85:3264-3273.
7. Frey T. 1997. Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28:253-263.
8. Homburg C.H., M. de Hass, A.E. von dem Borne, A.J. Verhoeven, C.P. Reutelingsperger and D. Ross. 1995. Human neutrophils lose their FcgRIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 85(2):532-540.
9. Horvatinovich J.M., S.D. Sparks, and M.J. Borowitz. 1994. Detection of terminal deoxynucleotidyl transferase by flow cytometry: a three color method. Cytometry (CCC) 18:228-230.
10. Ormerod M.G. 1994. Further application in cell biology. In: Flow Cytometry: A Practical Approach, Second Edition, M.G. Ormerod (Ed), IRL at Oxford University Press, pp. 261-273.
11. Renò F., E. Falcieri, F. Luchetti, S. Burattini and S. Papa. 1997. Discrimination of apoptotic cells in flow cytometry using Trypan Blue and FDA. Eur. J. Histochem. Vol. 41 Suppl.2:115-116.
12. Schmid I., C.H. Uittenbogaart and J.V. Giorgi. 1994. Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15:12-20.
13. Swat W., K Ignatowicz, and P. Kisielow. 1991. Detection of apoptosis in immature CD4+8+ thymocytes by flow cytometry. J.Immunol. Methods 137:79-87.
14. Weber G.F., J. Daley, S.K. Kraeft, L.B. Chen, and H. Cantor. 1997. Measurement of apoptosis in heterogeneous population. Cytometry 27:136-144.
15. Wu X.M., Z. Ao, M. Hegen, C. Morimoto and S.F. Schlossman. 1996. Requirement of Fas (CD95), CD45, and CD11a/CD18 in monocyte-dependent of human T cells. J.Immunol.157:707-713.

RPMI 1640 supplemented with 10% heat inactivated fetal calf serum (FSC), Penicillin/Streptomycin 1000U/1000µg/ml, 200 mM Glutamine, Sodium piruvate 100 mM and buffered with NaHCO₃.

Phosphate buffer (PBS): 138 mM NaCl, 27 mM KCl, 1.76 mM KH₂PO₄, 8 mM Na₂HPO₄

Hepes buffer :10 mM Hepes/NaOH, pH 7.4, 150 mM NaCl; 5 mM Kcl, 1 mM MgCl₂, 1.8 mM CaCl₂. Store at 4°C, stable for 1 month.

Fluorescein diacetate (FDA): dissolved in acetone (1 mg/ml) and diluted in PBS at a concentration of 1µg/ml shortly before use. Store at 4°C and protect from light. Stable for at least 2 months.

Trypan Blue: dilute at 0.8 mM in PBS or Hepes. Store at room temperature. Stable for 1 month. Trypan Blue is considered a cancerogenic product .

Appendix 2: Reagents
 

Annexin V/Fitc Cod. BM S306FI
Bender-Medsystems

Camptothecin Cod. C9911
Sigma

CD11a Cod. 45195F / CBL451 PE
Eurobiox

FDA Cod. T-F-7378
Sigma

Trypan Blue Cod. T-8154
Sigma

Centrifuge Sorval, mod.MC12V, DuPont.

Epics XL cytometer, equipped with an air-cooled argon ion laser tuned at 488nm wavelength, cod.

Coulter Corporation, U.S.A..