The most widely used method for assessing apoptosis is the qualitative analysis of DNA fragmentation at internucleosomal sites by gel electrophoresis, combined with the measurement of low molecular weight DNA extractable from cells after mild lysis ¹. However, this approach suffers from important limitations such as its relatively low sensitivity and its inability to discriminate which cell types undergo apoptosis in mixed population. Several alternative methods² are based on the flow cytometric analisys of cellular light scattering and of the uptake of DNA-binding fluorescent dyes such as propidium iodide³_, Hoechst 33342^4,5_, ethidium bromide^6,7, 7-amino-actinomycin D (7-AAD)⁸, or other dyes⁹. However, these methods either cannot identify the phenotype of dead cells or cannot discriminate between apoptosis and necrosis. Furthermore, the permeability to propidium iodide and 7-AAD varies from the early to the late phases of cell death^2,5.

Apoptosis is accompanied by water loss, shrinkage of the cell, and enzymatic fixation of the membrane, whereas necrosis is not. Necrotic and apoptotic cells differ, therefore, both in differential light scattering and in the antigenic make-up of the cell membrane.

We describe a quantitative and sensitive flow cytometric method¹⁰ for the detection of human apoptotic lymphocytes that, unlike previously published assays, allows their identification in mixed populations of peripheral bloood leukocytes as well as their immunophenotyping. This method is based on integration of differential light scatter parameters (Forward Scatter vs Side Scatter in linear amplification) with apparent density of CD45 (dim vs bright in logaritmic amplification) membrane fluorescence. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased fluorescence intensity of surface CD45. Furthermore, apoptotic cells cen be electronically gated (CD45 intensity vs light scatter) and characterized phenotypically by tow or tree colour staining.
 

2.1 Cytometer setting. Flow cytometric analyses were performed using three different instruments: Cytoron Absolute with 256-channels resolution, FACScan and FACScalibur with 1024-channels resolution (A3). The differential light scatter detectors may be set to specifically focus on the lymphocyte region, partially sacrificing the contemporaneous vision of granulocytes and monocytes. This can be achieved by incrementing the amplification gain of the forward scatter (FW-SC) photodiode and both the amplification gain and the excitation voltage of the right scatter (RT-SC) photomultiplier.

 

2.2 Staining with MoAbs (basic protocol). For each determination, 5x10⁵ nucleated cells in 50 µl of RPMI 1640 with 10% fetal calf serum (complete medium, A2) are single, double or triple-stained with anti-CD45 fluorescein isothiocyanate (FITC) conjugated (A2) and different phycoerythrin (PE) or red emitting fluorocrome (e.g.: peridinchlorophyll protein, PerCP) conjugated MoAbs, at 4°C for 30 minutes, using saturating amounts of antibody experimentally determined for each MoAb. After one wash the cells are resuspended in complete medium and directly analyzed by flow cytometry or stained for membrane permeability (support protocol).

 

Staining with dyes (support protocol). Membrane permeability, as an indicator of late apoptosis and cell death, is evaluated by staining with propidium iodide (PI, A2), when cells are CD45-FITC single stained, and by 7-amino-actinomycin D (7-AAD, A2) when membrane double staining (CD45FITC/CD"X"PE) is performed. The intercalative dyes are used at 1 mg/ml in complete medium, incubated at 4°C for 30 minutes and analyzed without further washings.

 

2.3 Data analysis. On biparametric scattergram of 10,000 events, gated for CD45 positivity and acquired in list mode, are defined two different regions: one includes apoptotic cells with lower size and apparent greater complexity (low FW-SC and high RT-SC), and another includes events with differential light scatters (DLS) property typical of viable cells. The percentage of apoptotic cells is calculated on the basis of the number of CD45^dim cells in apoptotic region versus the total number of CD45⁺ cells in both regions. An objective boundary between CD45^dim and CD45^bright cells is determinated using Kolmogorov-Smirnov statistics on the overlayed CD45 fluorescence intensity histograms of the two regions, taking as the cut-off point the fluorescence channel with maximum difference between the two histograms.

The same procedure can be applied on cells belonging to defined subpopulations identified by staining with antisubset antibodies in combination with anti-CD45. In these cases a logical gate including scatter region, CD45 and a given subset marker must be defined. In the same way is possible to evaluate the membrane permeability by intercalative dyes, taking account that PI is compatible with FITC fluorescence only while 7-AAD can be used with FITC and PE stained cells.
 

 
3.1 Background Information.

To obtain a good performance using this assay two parameters are critical. A daily check up of the cytometer alignment and fluidic, must be done using standard microbeads to verify the scatter and fluorescence coefficient of variation (CV).

The other critical parameter is the CD45 histogram sharpness. In our hands FITC conjugated MoAbs to leukocyte common antigen showed best results. The reactive quality can be evaluated by the resolution degree of CD45 intensity histograms of lymphocytes, granulocytes and monocytes.

3.2 Key References.

Anti-CD45: FITC cod. 347463 Becton-Dickinson, San Jose CA

Fetal Calf Serum: Biochrom KG, Berlin, Germany

RPMI 1640: Gibco, Grand Island, NY

PI: cod. P-1304; Molecular Probes Inc, Eugene, OR

 
Appendix 2: Equipment
Cytoron Absolute: Ortho Diagnostic Systems, Raritan, NJ

FACScan: Becton Dickinson, San Jose, CA

FACScalibur: Becton Dickinson, San Jose,CA