Current Protocols in Immunology

This Featured Protocol presents a cutting-edge method excerpted from Current Protocols in Immunology UNIT 3.17.

Loss of viability, whether the result of a necrotic or apoptotic process, is often defined as loss of membrane integrity. Necrosis refers to the morphology usually associated with accidental cell death, while apoptosis is seen when cell death is programmed or physiologically regulated. The determination of whether a cell dies by apoptosis as opposed to necrosis is best made on the basis of distinct structural changes in the cell's chromatin, which occur prior to the lysis of the membrane. These changes include extensive condensation of the chromatin, as assessed by light or electron microscopy, and DNA fragmentation, as detected by sedimentation assays, gel electrophoresis, or end labeling of the DNA fragments. Loss of membrane integrity is conveniently measured by uptake of certain dyes such as trypan blue (APPENDIX 3), eosin, ethidium bromide, or propidium iodide (UNIT 5.4), or by release of radioactive chromium (UNIT 3.11) or lactate dehydrogenase (UNIT 3.16).

The first five protocols presented here are based on DNA fragmentation. Basic Protocol 1 describes the use of DNA-binding fluorescent dyes to determine the percentage of cells undergoing apoptosis and/or dying in a given population. Basic Protocol 2 and Alternate Protocols 1 and 2 cover assays for quantifying DNA fragmentation by centrifugal sedimentation. Finally Basic Protocol 3 outlines a simple method to assess DNA fragmentation qualitatively using agarose gel electrophoresis. Support Protocols 1 and 2 describe methods to radiolabel the DNA and the cytoplasm of the cells to be tested.

Another technique for detecting apoptotic cells is flow cytometry. Basic Protocol 4 describes a flow cytometric assay for quantifying viable cells, and Alternate Protocol 3 describes another flow cytometric technique that has the powerful advantage of measuring the cell loss of a subpopulation of cells defined by antibody labeling. Support Protocols 3 and 4 describe methods for priming T cell clones and freshly isolated lymph node cells, respectively, for T cell receptor (TCR)-induced apoptosis.

End labeling to detect DNA fragmentation is a more recently developed technique for identifying apoptotic cells. Basic Protocols 5 and 6 are based on the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method, which detects apoptotic cells by using the enzyme terminal deoxynucleotidyl transferase (TdT) to directly label the ends of broken DNA strands. This method is suitable for both quantitative and qualitative analysis; Basic Protocol 5 outlines a method for flow cytometric quantitation of apoptotic cells using TUNEL and Basic Protocol 6 describes a method for TUNEL staining of tissue sections to identify apoptotic cells.

Because the mechanism of apoptosis is poorly understood at the present time, it is probably best to perform several of the basic protocols to confirm an observation of apoptotic cell death. In addition, it should be noted that these assays have been used primarily in systems employing normal and tumor cells of murine hematopoietic origin; it has not yet been established whether cells of other origins or species undergo apoptosis by the same mechanism (see Commentary and Table 3.17.1).

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique must be used accordingly. BASIC PROTOCOL 5 FLOW CYTOMETRIC QUANTITATION OF APOPTOTIC CELLS USING TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) is a method for detecting apoptotic cells that exhibit DNA fragmentation. TUNEL (Gavrieli et al., 1992; Gorczyca et al., 1993) involves end labeling the broken ends of the double-stranded DNA with biotin-conjugated dUTP using the enzyme terminal deoxynucleotidyl transferase (TdT). This protocol outlines a method for quantitating TUNEL-stained apoptotic cells by flow cytometric analysis. TUNEL staining is more sensitive than other methods (i.e., PI staining of unfixed cells or PI definition of subdiploid DNA). In addition, it allows apoptotic cells to be analyzed individually on a flow cytometer and is compatible with simultaneous multicolor cell-surface staining, which permits quantitation of cell death in specific subpopulations of cells that are discernible only by means of multicolor flow cytometric analysis (Kishimoto et al., 1995). If desired, this TUNEL protocol can be performed immediately following (but not before) cell-surface marker staining (UNIT 5.3). Materials

Cells for analysis

PBS (APPENDIX 2)

95% ethanol (not 100% ethanol), ice cold

Paraformaldehyde fixative (UNIT 4.7)

TdT reaction buffer (see recipe)

TdT/biotin-dUTP mix (see recipe)

Fluorescein isothiocyanate (FITC)-conjugated streptavidin (Jackson Immunoresearch; follow manufacturer's instructions for appropriate dilution)

12×75-mm round-bottom centrifuge tubes

IEC 6R6000 centrifuge with model 269 rotor (or equivalent)

Additional reagents and equipment for immunofluorescence staining (optional; UNIT 5.3) and flow cytometric analysis (UNIT 5.4)

1. a. For multicolor analysis: Perform immunofluorescence staining (UNIT 5.3). Resuspend stained cells in PBS and transfer an aliquot containing 5×10⁵ cells to a 12×75-mm round-bottom centrifuge tube.
    

   If this step is performed, it must be done prior to the TUNEL procedure. The final wash should be performed in PBS (instead of the staining buffer used in UNIT 5.3) in 12×75-mm round-bottom centrifuge tubes and the cells should not be treated with propidium iodide. It is essential that the fluorochromes used for multicolor staining not be destroyed by the fixation steps used in the TUNEL procedure. FITC, PE, and Texas red are not affected. The duochromes (conjugates of PE and Texas red--e.g., Red 613) are not affected either, but allophycocyanin (APC) is destroyed by the fixation.

1. b.For TUNEL detection alone: Transfer an aliquot containing 5×10⁵ cells to a 12×75-mm round-bottom centrifuge tube.
    
2. Add 1 to 2 ml PBS, then centrifuge cells 5 min at 300×g (1200 rpm in an IEC model 269 rotor), 4°C, and decant supernatant. Resuspend pellet in 250 µl PBS, then add 750 µl ice-cold 95% ethanol dropwise over a period of 5 to 10 sec while gently vortexing. Incubate 20 min at 4°C.
    

   Gradual addition of ethanol while gently vortexing reduces clumping during this fixation step.
    

3. Wash cells by adding 2 ml PBS and centrifuging 5 min at 400×g (1500 rpm in an IEC model 269 rotor), 4°C. Decant supernatant.
    

   Higher centrifugation speed is required for fixed cells than for unfixed cells because ethanol fixation causes cell shrinkage.
    

4. Flick the tube to resuspend cells in residual buffer and add 1 ml paraformaldehyde fixative dropwise over a period of 5 to 10 sec while gently vortexing cells. Incubate 30 min at room temperature.
    

   Paraformaldehyde fixation makes the intracellular constituents more accessible and greatly increases the sensitivity of TUNEL staining.
    

5. Wash cells with PBS as in step 3.
    

   The washed cells can be stored for a few days at 4°C in the dark.
    

6. Wash cells as in step 3, using 0.5 ml TdT reaction buffer in place of PBS. Remove as much supernatant as possible by touching the lip of the inverted tube on absorbent paper immediately after decanting the supernatant.
    
7. Add 50 µl TdT/biotin-dUTP mix to cell pellet. Incubate 45 min at 37°C.
    
8. Wash cells with PBS as in step 3, then add 10 µl FITC-conjugated streptavidin (at the dilution recommended by the manufacturer). Incubate 30 min at room temperature, then wash again with PBS as in step 3.
    

   The best staining is obtained with FITC-conjugated streptavidin. Other fluorochromes--e.g., PE and the "duochromes"--give much weaker staining. This is probably because the higher-molecular-weight fluorochromes do not penetrate efficiently into the fixed cells.
    

9. Perform flow cytometric analysis (UNIT 5.4).
    

   Results for representative histograms obtained from flow cytometry using the TUNEL method.See Anticipated

Fresh tissues for analysis

1% (w/v) paraformaldehyde in PBS (dissolve by stirring with low heat overnight and filter before use)

Tris-buffered saline (TBS; APPENDIX 2)

0.1% (v/v) H₂O₂ in TBS

TdT reaction buffer (see recipe)

TdT/digoxigenin-dUTP mix (see recipe)

2% (v/v) horse serum or FBS in TBS

Sheep anti-digoxigenin primary antibody solution (see recipe)

HRPO-conjugated anti-sheep secondary antibody solution (see recipe)

AEC substrate working solution (see recipe)

Mayer's hematoxylin (Sigma)

Crystal Mount mounting medium (Fisher)

Hydrophobic-barrier slide marker (e.g., PAP Pen; Research Products International)

Coplin jars or staining trays

Humidified container (see recipe)

Additional reagents and equipment for preparing frozen sections (as in immunoperoxidase staining; UNIT 5.8)

1. Prepare 5- to 8-µm frozen sections (see UNIT 5.8 Basic Protocol, steps 1 to 7), drying slides overnight at room temperature.
    

   Slides can be stored 2 to 3 weeks at 4°C or a few months at -70°C. This prolonged storage is possible because DNA is fairly stable. Paraffin sections may also be used; these should be prepared, rehydrated, and air dried as in UNIT 5.8 Alternate Protocol, steps 1 to 10 (omitting step 8).
    

2. Draw a hydrophobic boundary on the glass around each section with a PAP Pen.
    

   A tight boundary is critical because each section must be covered with a small volume of TdT reaction buffer (see step 7).
    

3. Generously cover the entire section with 1% paraformaldehyde and incubate 30 min at room temperature in a closed container.
    

   A closed container is necessary to prevent evaporation but a humidified container is not required.
    

   Sections can be fixed with acetone instead of paraformaldehyde for 5 min at room temperature. Acetone-fixed sections tend to stain with better resolution, but such staining is generally weaker and tends to have a higher background. If the sections are to be acetone fixed, draw the boundaries with the PAP Pen after the acetone has completely evaporated.

4. Pour off the paraformaldehyde and wash the slide by incubating 5 min at room temperature in a Coplin jar or staining tray containing TBS, dipping the slide in and out of the solution three to four times during the course of the incubation.
    

   This step is not required for acetone-fixed sections.
    

5. Cover section with 0.1% H₂O₂ in TBS. Incubate 30 min at room temperature in a closed container to quench endogenous peroxidase activity.
    

   For acetone-fixed sections use 0.01% H₂O₂.
    

6. Wash with TBS as in step 4, then cover section with TdT reaction buffer to rinse out TBS.
    

   This step is critical because the TdT reaction is highly sensitive to buffer conditions.
    

7. Remove as much reaction buffer as possible and add 25 µl TdT/digoxigenin-dUTP mix. Incubate 45 to 60 min at 37°C in a humidified container.

8. Wash slide once with TBS, then once with 2% horse serum or FBS in TBS, each time using the washing technique described in step 4.
    
9. Cover section with sheep anti-digoxigenin primary antibody solution and incubate 1 hr at room temperature in a closed container.
    
10. Wash slide as in step 8, then cover section with HRPO-conjugated anti-sheep secondary antibody solution and incubate 1 hr at room temperature in a closed container.

11. Wash slide as in step 8, then cover section with AEC substrate working solution and incubate 10 to 20 min at room temperature.
     

    The intensity of color development can be visually monitored by low-power light microscopy. Develop until positive staining is strong with a minimal background.
     

    Diaminobenzidine (DAB) may be used as a substrate in place of HRPO (see UNIT 5.8). This helps avert one critical drawback in using AEC--i.e., that the color precipitate starts to fade slowly within a few weeks. Although fading is not a problem with DAB, sections developed with this substrate have less resolution because DAB tends to diffuse.
     

12. Wash slide as in step 8, then counterstain by incubating 0.5 to 1 min in Mayer's hematoxylin, then washing 5 min with tap water in a Coplin jar.
     

    This step is optional; omitting it greatly reduces the fading problem with AEC staining. Where sections are not counterstained, the AEC-stained slide (from step 11) should be washed as in step 8; the coverslip should then be mounted aas in step 13.
     

13. Wipe excess water from around the section and mount coverslip with Crystal Mount.
     

    See Anticipated Results and Figure 3.17.4 for representative stainings obtained using in situ TUNEL.
     

    Because of the fading problem with AEC, sections developed with this reagent should be photographed within a few days after staining.

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    Table 3.17.1 Apoptosis Studies in Various Cells of the Immune System^a

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    Cell type	Treatment	DNA cleavageb	Assay time (hr)	Apoptotic indexc	Protein synthesis requiredd
    Mouse
    Thymocytes	100 nM dexamethasone	DS-L	6	40-60	Yes
    		DS-L	24	80-100	Yes
    	600-rad -irradiation	DS-L	6	40-60	Yes
    		DS-L	24	80-100	Yes
    	43ºC for 60 min	DS-L	6	40-60	Yes
    		DS-L	24	80-100	Yes
    	1 µCM A23187	DS-L	24	40-60	Yes
    	Anti-CD3	DS-L	24	20-60	Yes
    	--	--	24	10-30	Yes
    Lymph node T cell	600-rad -irradiation	DS-L	24	90-100	Yes
    	43ºC for 60 min	DS-L	24	40-60	?
    S49.1	100 nM dexamethasone	DS-L	24	10-50	Yes
    	43ºC for 60 min	DS-L	24	30-50	?
    CTLL-2	Removal of IL-2	DS-L	24	50-100	Yes
    	Cytotoxic T cells	DS-L	4	50-100	No
    P815	Cytotoxic T cells	DS-L	4	50-100	No
    	--	--	4	5-15	--
    3T3	Cytotoxic T cells	SS-N	8	0-10	No
    Human
    HL-60	43ºC for 60 min	DS-L	24	10-50	No
    Raji	Cytotoxic T cells	DS-R	4	10-20	No

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    ^a See text (background information) for further description of cell types and treatments employed in these studies.

    ^b Types of DNA cleavage observed: DS-L, double-stranded cleavage in the linker region between nucleosomes as detected by agarose gel electrophoresis (see third basic protocol); SS-N, extremely rare single-stranded nicks as detected by alkaline sucrose-gradient centrifugation; DS-R, rare (every 50 to 100 kb) double-stranded breaks as detected by neutral sucrose-gradient centrifugation.

    ^c Apoptotic index refers to the expected values for percent fragmented DNA at the indicated times as quantified according to the first and second basic protocols.

    ^d Requirement of protein synthesis refers to whether inhibitors of RNA (e.g., actinomycin D) or protein (e.g., cycloheximide, emetine, and pactamycin) synthesis prevent apoptosis (nuclear damage as well as cell lysis).

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