Method Booklet 3. Apoptosis Applications & Glossary

Transcription

1 Method Booklet 3 Apoptosis Applications & Glossary

2 Apoptosis Applications & Glossary Table of Contents Introduction Annexin-V Caspases Fluorometric detection Colorimetric detection PARP BID DNA Fragmentation TUNEL Appendix: other apoptosis products References Glossary

3 Introduction Apoptosis, defined as programmed cell death, plays a very important role in many physiological and pathological conditions such as embryo and organ development, immune responses, tumor development and growth. Detecting apoptotic cells or monitoring the cells progressing to apoptosis is an essential step in basic research and in developing drugs that may regulate apoptosis. Apoptosis is characterized by many biological and morphological changes; such as, change of mitochondrial membrane potential, activation of caspases, DNA fragmentation, membrane blebbing and formation of apoptotic bodies. Based on these changes, various assays are designed to detect or quantitate apoptotic cells. Typical assays include Annexin- V binding, caspase enzyme activity, TUNEL (terminal deoxynucleotidyl transferase-mediated dutp nick-end-labeling) and DNA gel electrophoresis (see Table 1, next page). The goal of this manual is to provide customers with proven methods that highlight the menu of products offered by BioSource International, Inc. Our Research and Development staff has detailed the protocols used to obtain the results given here. When appropriate, buffer formulations and specific parameters for instruments are also provided. As always, your feedback has contributed to the initiative for this publication and will continue to affect improvements in the materials which are supplied with our products. Please contact our Technical Service staff with any inquiries regarding this manual. (tech.support@biosource.com or ) The first method which is described in this publication is the detection of Annexin V-FITC by flow cytometry. This method has become a hallmark for the separation of necrotic cells from those undergoing true apoptosis. BioSource International, Inc. has formulated a kit containing Annexin V-FITC and propidium iodide, along with detailed instructions on how to distinguish cells in early apoptosis, late apoptosis, and necrosis, utilizing a two- color staining regime. Quantitation of caspase activity is another popular means of determining what stage of apoptosis may be occurring. Caspases are protein-cleaving enzymes which are instrumental in the sequential disassembly of cells. BioSource International, Inc. has formulated kits for screening cell lysates for Caspase-2, -3, -6, -8, and -9 activity by either fluorometric or colorimetric detection. Also available are antibodies for Caspase-1, -3, -6, -8 and -10 for use in Western blotting. A novel method for detection of apoptosis by flow cytometry is the PARP-FITC Cleavage Site-Specific Antibody (CSSA). This FITC-conjugated anti-parp antibody specifically recognizes the 85kDa fragment of cleaved PARP which can be an excellent marker for detecting apoptosis. Along with the PARP-FITC CSSA, BioSource International, Inc. also provides an unconjugated and Biotin form of the antibody, and has formulated a PARP-FITC CSSA Kit which includes fixation and permeabilization buffers and matching peptide. Cleaved BID (BH3 interacting domain death agonist) plays an important role in the transduction and amplification of apoptotic signals during apoptosis. BioSource International, Inc. offers four BID antibodies that detect full length BID in both human and mouse, a BID p15 fragment that recognizes human BID, and a BID [59/60] CSSA that specifically recognizes cleaved mouse BID. DNA fragmentation occurs when nuclear enzymes become active and destroy the normal chromatin structure of DNA. The DNA becomes fragmented in pieces of 200 bp or less in length. This fragmentation can easily be visualized by agarose gel. BioSource International, Inc. provides a simple Quick Apoptotic Ladder Kit for determining if fragmentation of DNA has occurred, allowing one to distinguish between apoptotic cells and normal cells. Breaks in the DNA can be further distinguished by use of bromodeoxyuridine triphosphate (Br-dUTP). Br-dUTP incorporates into breaks in the DNA. One can then label the broken DNA with an antibody to Br-dUTP as provided in the APO-BRDU TUNEL kit to distinguish apoptotic cells from normal and necrotic cells by flow cytometry. A wide variety of additional reagents for apoptosis research are described in the appendix including antibodies, ELISA kits, mrna kits, primers, substrates and inhibitors. Table 1: Comparison of Annexin-V assay with Caspase-3 and TUNEL assay Assays Annexin-V binding assay Caspase-3 assay TUNEL assay Mechanism Apoptotic cells have PS DEVD-AFC (fluorometric) or Exogenous TdT incorpoexposure. Annexin-V DEVD-pNA (colorimetric) rates dutp to the ends of conjugates bind to PS on is used as substrate to detect DNA fragments, which the cell surface and Caspase-3 and Caspase-3 like can be detected with identify apoptotic cells. enzyme activity. various methods. Stage of apoptosis Relatively early, but Early. Late. after caspase activation. Adherent cells or Useful for suspension cells Useful for adherent cells and Useful for adherent cells or suspension cells and difficult to perfrom on suspension cells. suspension cells. adherent cells. Tissues Not recommended. Not recommended. Can be used on tissue sections. Sample storage Assay done immediately Treated samples may be stored Treated cells may be fixed following the induction at 20 C for further assay. and stored. of apoptosis. Multiple color Can be used for multiple May not be used with other Potential multiple color analysis color analysis and markers. analysis. Analyzes analyzing apoptosis at single apoptotic cells at single cell level. cell level. Time consuming Simple procedure and short Several procedures and Multiple procedures and and procedures incubation, 15 minutes. long incubation (1-2 hours). multiple washing needed. Number of samples More samples (decades) Many samples (hundreds) Limited samples can be can be handled can be done in one can be done in one done in one experiment. experiment. experiment. Instrument Flow cytometer or Spectrofluorometer or spectro- Flow cytometer, fluorfluorescence microscope. photometer (microtiter plate escence or light microscope. reader). Non-specific Potentially high non- Some background on non- Low background on nonbackground specific background if the apoptotic cells (untreated). apoptotic cells. cell culture condition is not optimal because of dead cells.

4 We welcome your feedback on the methods described herein. Please contact us with your comments. All methods and products are for research purpose only and not intended for diagnostic use. Disclaimer: The procedures presented in this manual are accurate to the best of our knowledge. BioSource International, Inc. makes no warranties either expressed or implied as to any matter whatsoever, including, without limitations, the condition of the products, their merchantability, or fitness for any particular use. BioSource International, Inc., shall not be held liable for any direct, indirect, incidental, or consequential damages, including without limitation, loss of profit, loss of business, or other loss which may be based directly or indirectly upon the sale, use of products, or inadequacy of product for any purpose or by any defect or deficiency therein, even if BioSource International, Inc., knew or should have known of the possibility of such loss. The products listed in this booklet are for research use only, and are not intended for use in diagnostic procedures. Chapter 1: Annexin-V Introduction The Annexin-V binding assay is based on the relocation of phosphatidylserine to the outer cell membrane. Viable cells maintain an asymmetric distribution of different phospholipids between the inner and outer leaflets of the plasma membrane. Choline-containing phospholipids such as phosphatidylcholine and sphingomyelin are primarily located on the outer leaflet of viable cells and aminophospholipids such as phosphatidylethanolamine and phosphatidylserine (PS) are found at the cytoplasmic (inner) face of viable cells. The distribution of phospholipids in the plasma membrane changes during apoptosis. In particular, PS relocates from the cytoplasmic face to the outer leaflet so called PS exposure. The extent of PS exposure can distinguish apoptotic cells from the non-apoptotic cells. Annexin-V is a kda calcium-dependent phospholipid binding protein with high affinity for PS (kda ~ 5x10-10 M). When labeled with a fluorescent dye, Annexin-V can be used as a sensitive probe for PS exposure on the outer leaflet of the cell membrane. The binding of Annexin-V conjugates such as Annexin-V FITC to cells permits differentiation of apoptotic cells (Annexin-V positive) from non-apoptotic cells (Annexin-V negative) (see Figure 1, page 6). Annexin-V binding is observed under two conditions. The first condition is observed in cells midway through the apoptosis pathway. Phosphatidylserine translocates to the outer leaflet of the cell membrane. The second condition is observed in very late apoptosis or when the cells become necrotic and membrane permeabilization occurs. This membrane permeabilization allows Annexin-V to enter cells and bind to phosphatidylserine on the cytoplasmic face of the membrane. Since other causes besides apoptosis can result in necrosis, it is important to distinguish between necrotic and apoptotic cells. Membrane permeabilization also permits entry of other materials to the interior of the cell, including the fluorescent DNA-binding dye propidium iodide. Utilizing dual staining methodology, apoptotic populations can be distinguished from necrotic populations. For example, using the Annexin V-propidium iodide (PI) double staining regime, three populations of cells are distinguishable in twocolor flow cytometry (Figure 1). Non-apoptotic cells: Early apoptotic cells: Necrotic cells or late apoptotic cells: Annexin-V negative and PI negative; Annexin-V positive and PI negative; Annexin-V positive and PI positive. In summary, Annexin-V FITC/PI dual staining is very useful for defining apoptotic suspension cells by flow cytometry.

5 Typical Data Using Annexin-V FITC Kit A Propidium Iodide B 2. Dilute 10x Annexin-V Binding Buffer 1:10 in distilled water. 3. When apoptotic induction of cells is complete, wash the cells with PBS twice and resuspend cells at 2-3 x 10 6 cells/ml in 1x Annexin-V Binding Buffer. 4. Aliquot cells at 100 µl/tube. 5. Add 5 µl of Annexin-V FITC and 10 µl of Propidium Iodide Buffer to each tube. 6. Incubate at room temperature for 15 minutes in the dark. 7. Add 400 µl 1x Annexin-V Binding Buffer to each tube. 8. Analyze the cells by flow cytometry within 1 hour of staining. Annexin-V Troubleshooting Guide Figure 1: Jurkat cells were cultured with 2 µm camptothecin for 5 hours and stained by Annexin-V FITC (Catalog #PHN1008). Apoptotic and non-apoptotic cells can be differentiated according to Annexin-V FITC binding (A). M1 represents non-apoptotic cells and M2 represents Annexin-V positive cells. Staining with propidium iodide (Catalog #PNN1011) differentiates the Annexin-V positive population (apoptosis) from cells in necrosis (B). Conclusion: propidium iodide stained cells are easily distinguished from early apoptotic population (Annexin-V positive and PI-negative) appearing in the lower right quadrant of the two color dot plot. Method Annexin-V FITC Log Green Fluorescence Annexin-V FITC Purpose: The ApoTarget Annexin-V FITC Apoptosis Kit is designed to detect apoptotic cells by flow cytometry or immunofluorescence. The ApoTarget Annexin-V FITC Apoptosis Kit (Catalog #PHN1010) employs a fluorescein-labeled Annexin-V (Annexin-V FITC) in concert with propidium iodide to detect the cells undergoing apoptosis. Reagents and Equipment 1. Annexin-V FITC (Catalog #PHN1008) 2. Propidium Iodide Buffer (Catalog #PNN1011) 3. Annexin-V Binding Buffer (Catalog #PNN1001) 4. Fluorescence activated cell sorter with a laser using excitation at 488 nm (for detection by flow cytometry) 5. PBS 6. Calibrated adjustable precision pipettes, preferably with disposable plastic tips 7. Tubes appropriate for holding cells during induction of apoptosis 8. Microcentrifuge Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. Problems Causes Solutions High background on 1. Cell culture condition 1. Change cell culture condition, use non-stimulated cells may be not optimal. lower cell density. 2. Cells have been stored too 2. Perform staining immediately after long after the treatment. collecting cells. 3. Cells have high constitutive 3. Use a lower amount of Annexin-V PS on cell surface. conjugates per test. Low percentage of 1. Inducers are not effective. 1. Use camptothecin to treat Jurkat apoptotic cells cells as positive control to check Annexin-V conjugates and then use optimal induction conditions. 2. Cell culture condition may 2. Grow cells in optimal condition. not be optimal, e.g., overgrown cells may be less sensitive to stimuli. 3. Annexin-V Binding Buffer 3. Use freshly prepared Annexin-V is not effective. Binding Buffer. 4. Analyzed stained cells too 4. Analyze cells within one hour long after staining. after staining. There is an intermediate 1. PI concentration is too 1. Reduce the concentration of PI stained population high for the cells. Propidium Iodide. Product Catalog # Size Annexin-V FITC Kit PHN Tests Annexin-V FITC Kit PHN Tests Annexin-V Biotin PHN Tests Annexin-V Cy3 PHN Tests Annexin-V FITC PHN Tests Annexin-V Binding Buffer PNN ml Propidium Iodide Buffer PNN ml

6 Chapter 2: Caspases Introduction One group of cysteinyl-aspartic acid proteases is called caspases. Caspases are members of the Interleukin-1β Converting Enzyme (ICE) family of cysteine proteases. There are 14 caspases that have been identified which include: Caspase-1 Caspase-2 Caspase-3 Caspase-4 Caspase-5 Caspase-6 Caspase-7 Caspase-8 Caspase-9 Caspase-10 Caspase-11 Caspase-12 Caspase-13 Caspase-14 Also known as ICE Also known as Ich-1, Nedd2 Also known as CPP32, Yama, Apopain, SCA-1, and LICE Also known as ICEreI-II, TX and ICH-2 Also known as ICErel-III and TY Also known as Mch-2 Also known as Mch-3, ICE-LAP-3 and CMH-1 Also known as FLICE, Mach-1 and Mch5 Also known as ICE-LAP6, Mch6 and Apaf-3 Also known as FLICE-2, Mch4 Also known as ERICE Also known as Mini-ICE Through the use of synthetic peptides, Caspases have been divided into three groups based on the four amino acids amino-terminal to their cleavage site. Caspases-1, -4 and -5 prefer substrates containing the sequence WEXD (where X is variable). Caspases-2, -3 and - 7 prefer the sequence DEXD. Caspases 6, 8 and 9 are the least demanding but have demonstrated a preference for cleaving of substrates containing either LEXD or VEXD. Because these sequences correspond to known cleavage sites of caspase targets, systems to study caspase cleavage activity have been developed. The measurement of caspase enzyme activity with fluorometric and colorimetric peptide substrates and the detection of caspase cleavage using antibodies to caspases allows the study of the apoptosis processes or screening of therapeutic agents which promote or prevent apoptosis. BioSource International, Inc. provides Caspase-2, -3, -6, -8 and -9 protease activity assay kits (both fluorometric and colorimetric) as well as antibodies specific for Caspase-3,-6,-8 and -10. Figure 1: Phylogenetic analysis of caspases reveals that there are four subfamilies: Subfamily Caspase ICE subfamily Caspase-1, -4 and 5 Ced-3/CCP32 subfamily Caspase-3,-6, -7 Mach/FLICE subfamily Caspase-8, and 10 ICH (ICE and CED-3 homologue) subfamily Caspase-2 and -9 The caspases are synthesized as inactive pro-enzymes or pro-caspases. In apoptosis, the pro-caspases are processed by proteolytic cleavage to form active enzymes. For example, Caspase-3 exists in cells as an inactive 32 kda proenzyme, called pro-caspase-3. Pro- Caspase-3 is cleaved into active 17 and 12 kda subunits by upstream proteases to become active Caspase-3. Caspases-2, -8, -9 and -10 are classified as signaling or upstream (see Figure 1, next page) in the apoptosis pathway because long prodomains allow association with cell surface receptors such as FAS (CD95), TNFR-1 (CD120a), DR- 3 or CARD domains. This observation suggests a proteolytic cascade as a mechanism for signaling. A proteolytic cascade exists that would activate the terminal event required for apoptosis in a way similar to that of the coagulation cascade seen with the closely related family of serine proteases. For example, Caspase-4 activates pro-caspase-1; Caspase- 9 activates pro-caspase-3; and Caspase-3 cleaves pro-caspase-6 and pro-caspase-7. Caspases play a critical role in the execution phase of apoptosis. Important targets of caspases include cytoplasmic and nuclear proteins such as keratin 18, poly ADP ribose polymerase (PARP) and lamins. Overexpression of Caspase-3 induces apoptosis.

7 Methods for Detecting Caspase Activity Figure 2: The excitation and emission wavelengths for the fluorochromes of caspase substrates available from BioSource International, Inc. Excitation Emission AFC amino acid sequence of DEVD. The peptide substrate used would be DEVD with the D labeled with a colorimetric or fluorometric marker. The marker is then allowed to fluoresce or emit color once cleavage has occurred. Without cleavage, little or no signal should be observed. Thus, utilizing DEVD-AFC (Catalog #77-934), DEVD-AMC (Catalog #77-910) and MCA-DEVDARK[K-DNP] (Fluorescence Resonance Energy Transfer (FRET) peptide) (Catalog #77-966), cleavage of the peptide by the caspase can be observed. Figure 2 provides the information on the excitation and emission wavelengths for those fluorochromes of those caspase subsrates. See page 18 for a complete listing of caspase kits. Purpose: Following are two examples for in vitro determination of Caspase-3 proteolytic activity in lysates of mammalian cells using our simple and convenient caspase activity assay kits. Both methods provide a means for quantitating caspases that recognize the amino acid sequence, DEVD. One utilizes the fluorophore, AFC, for fluorometric analysis of samples, and the other is a colorimetric method utilizing pna as a marker. The kits include substrate and optimized buffers for the appropriate dye. Caspase Protease Activity Assay Induction of apoptosis in cells AMC Protease activation DEVD-AFC DEVD-pNA FRET Caspase-3 (CPP32) DEVD DEVD Wavelength (nm) Caspase proteins cleave other proteins after aspartic acid. From previous studies we know that the three to four amino acids prior to the aspartic acid confer specificity to the caspase. This allows the use of four amino acid-labeled peptides to be utilized as substrates for the caspases. For example, the clevage site for Caspase-3 corresponds to the AFC pna

8 Fluorometric Method: Caspase-3 The substrate, DEVD-AFC, is composed of the fluorophore, AFC (7-amino-4-trifluoromethyl coumarin), and a synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid sequence of the Caspase-3 cleavage site in PARP. DEVD-AFC emits blue light (λ max = 400 nm). Upon cleavage of the substrate by Caspase-3 or related caspases, with the excitation wavelength set to 400 nm, free AFC emits a yellow-green fluorescence (λ max = 505 nm) which can be quantified using a spectrofluorometer or a fluorescence microtiter plate reader. Comparison of the fluorescence of AFC from apoptotic samples with an uninduced control allows determination of the increase in Caspase-3 activity. Reagents and Materials 1. Cell Lysis Buffer 2. 2x Reaction Buffer 3. Substrate DEVD conjugated to the fluorophore AFC (Catalog #77-934) 4. 1M DTT 5. Fluorometer (and cuvettes) or fluorescent microplate reader equipped with excitation filter nm and emission filter nm. (maximal ex = 400 nm and em = 505 nm) (See figure 2) 6. Calibrated adjustable precision pipettes, preferably with disposable plastic tips 7. Protein measurement method, such as Bradford protein assay 8. Tubes appropriate for holding cells during induction of apoptosis 9. Microcentrifuge 10. Reaction tubes or 96-well microplate Procedure Summary for Caspase Assays (Fluorometric) Collect cells and resuspend in Lysis Buffer 10 minutes on ice Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. When the induction is complete, count cells and pellet 1-5 x 10 6 cells or use µg cell lysate, if protein concentration has been determined. 3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 minutes. 4. Prepare reaction buffer: Number of Samples Amount of 2x Reaction Buffer Amount DTT (sample x 50 µl) (10 µl x 2x vol.) µl 2.5 µl µl 5 µl ml 12.5 µl Determine the number of samples to be measured and aliquot enough 2x Reaction Buffer into a glass tube (assuming 50 µl of 2x Reaction Buffer per sample). Add DTT to the 2x Reaction Buffer immediately before use (10 mm final concentration: add 10 µl of 1.0 M DTT stock per 1 ml of 2x Reaction Buffer). 5. Add 50 µl of 2x Reaction Buffer (containing 10 mm DTT) to each sample. 6. Add 5 µl of the 1 mm DEVD-AFC substrate (50 µm final concentration) and incubate at 37 C for 1-2 hours. Keep the samples in the dark during incubation. 7. Read sample in a fluorometer or fluorescent microplate reader with a 400 nm excitation filter and 505 nm emission filter. 8. Fold-increase in Caspase-3 activity should be determined by direct comparison to the level of the uninduced control. Typical Data Using Fluorescent Caspase-3 Activity Assay Kit (Cat.# KHZ0012) The following absorbance data were obtained from Jurkat cells. Apoptosis was induced by incubating Jurkat cells with 0.2 µg/ml of anti-fas monoclonal antibody (Cat.# AHS9552) for 8 hours. The assay was performed according to the procedure described above. Figure 3: Add Reaction Buffer containing DTT Add Conjugated Protease Substrate 37 C for 1 hour Analyze using a Fluorometer λ ex=400 nm, λ em=505 nm

9 Caspase Fluorometric Assay Kit Troubleshooting Guide Problems Causes Solutions Unexpected high fluo- 1. Gain of fluorescence reader 1. Decrease gain. rescence development is too high. Colorimetric Method: Caspase-3 2. Too much protein. 2. Use less protein or dilute sample. Weak or no signal 1. Incubation of substrate in 1. Only use substrate solution containincubation buffer without DTT. ing DTT. 2. The caspase is digested 2. Perform lysis of cells on ice and nonspecifically. store at 20 C. 3. Induction of apoptosis is 3. Check inducers and conditions. not successful. 4. Unsuitable filters have 4. Check the filters for correct wavebeen used. length. 5. Gain of fluorescence reader 5. Increase gain. is too low. Poor precision 1. Non-homogeneous sample 1. Mix sample well before pipetting. after freezing. 2. Turbidity, particle or high 2. Centrifuge sample. lipid or DNA content within the sample. 3. Unequal volumes added 3. Check pipette function. to wells. The substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pna), and a synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid sequence of the Caspase-3 cleavage site in PARP. Upon cleavage of the substrate by Caspase-3 or related caspases, free pna light absorbance can be quantified using a spectrophotometer or a microplate reader at 400 or 405 nm. Comparison of the absorbance of pna from apoptotic sample with an uninduced control allows determination of the increase in Caspase-3 activity. Reagents and Materials 1. Cell Lysis Buffer 2. 2x Reaction Buffer 3. Substrate DEVD conjugated to the chromophore, pna (Catalog #77-900) 4. Dilution Buffer 5. 1M DTT 6. Spectrophotometer (and cuvettes) or microplate reader capable of measurement at nm 7. Calibrated adjustable precision pipettes, preferably with disposable plastic tips 8. Protein measurement method, such as Bradford protein assay 9. Tubes appropriate for holding cells during induction of apoptosis 10. Microcentrifuge 11. Reaction tubes or 96-well microplate Procedure Summary for Caspase Assays (Colorimetric) Collect cells and resuspend in Lysis Buffer Centrifuge and collect Supernatant Add Reaction Buffer containing DTT Add Conjugated Protease Substrate Analyze using a Spectrophotometer At 405 nm 10 minutes on ice 37 C for 1 hour Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. When induction is complete, count cells and pellet 3-5 x 10 6 cells per sample. 3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 minutes. 4. Centrifuge for 1 minute in a microcentrifuge (10,000 x g). 5. Transfer supernatant (cytosol extract) to a fresh tube and put on ice. 6. Assay protein concentration by any standard method. 7. Dilute each cytosol extract to a concentration of µg protein per 50 µl Cell Lysis Buffer (1-4 mg/ml). 8. Prepare Reaction Buffer: Determine the number of samples to be measured and aliquot enough 2x Reaction Number of Samples Amount of 2x Reaction Buffer Amount DTT (sample x 50 µl) (10 µl x 2x vol.) µl 2.5 µl µl 5 µl ml 12.5 µl

10 Buffer into a glass tube (assuming 50 µl of 2x Reaction Buffer per sample). Add DTT to the 2x Reaction Buffer immediately before use (10 mm final concentration: add 10 µl of 1.0 M DTT stock per 1 ml of 2x Reaction Buffer). 9. Add 50 µl of 2x Reaction Buffer (containing 10 mm DTT) to each sample. 10. Add 5 µl of the 4 mm DEVD-pNA substrate (200 µm final concentration) and incubate at 37 C for 2 hours. Keep the samples in the dark during incubation. 11. Read samples at 400 nm or 405 nm in a microplate reader, or spectrophotometer using a 100 µl micro-quartz cuvette, or dilute sample to 1 ml with Dilution Buffer and use a regular cuvette. Note: Dilution of the samples proportionally decreases the optical density. You may also perform the entire assay directly in a 96-well plate. 12. Increase in Caspase-3 activity should be determined by direct comparison to the level of the uninduced control. Uninduced cells Induced cells (non apoptotic) (apoptotic) Lysate (50 µl) x Reaction Buffer (50 µl) DEVD-pNA (5 µl) Control 1 Control 2 Control 3 Testing Calculation: Fold Increase = (Testing OD-Control 3 OD)/(Control 2 OD-Control 1 OD) Note: Background readings from cell lysates and buffers should be subtracted from the readings of both induced and uninduced samples before calculating fold-increase in Caspase-3 activity. Typical Data Using Colorimetric Caspase-3 Activity Assay Kit (Cat.# KHZ0022) The following absorbance data were obtained from Jurkat cells. Apoptosis was induced by incubating Jurkat cells with 0.2 µg/ml of anti-fas monoclonal antibody (Cat.# AHS9552) for 8 hours. The assay was done according to the procedure described above. Typically, the Caspase-3 activity in 20 µg-200 µg protein per sample can be detected. Figure 4: Caspase Colorimetric Assay Kit Troubleshooting Guide Problems Causes Solutions Weak or no signal 1. Incubation of substrate in 1. Only use substrate solution containincubation buffer without DTT. ing DTT. 2. The caspase is digested 2. Perform lysis of cells on ice and nonspecifically. store at 20 C. 3. Induction of apoptosis is 3. Check inducers and conditions. not successful. 4. Protein concentration is 4. Increase protein concentration. too low. 5. Inadequate incubation time 5. Increase incubation time. or temperature. High background on 1. Cell culture condition is 1. Check cell culture condition. non-apoptotic cells not optimal. Poor precision 1. Non-homogeneous sample 1. Mix sample well before pipetting. after freezing. 2. Turbidity, particle or high 2. Centrifuge sample. lipid or DNA content within the sample. 3. Unequal volumes added 3. Check pipette function. to wells. Limitations of the Procedure These kits provide a simple and convenient method to detect Caspase-3 activity of apoptotic cells. For fluorescent kits, if the reading of relative fluorescence is off scale high, the samples can be proportionally diluted with PBS. For both fluorescent and colorimetric assays, a relatively high concentration of DTT (10 mm) is required for full activity of the caspases. Make sure that DTT is added to the Reaction Buffer when the assay is carried out; otherwise, unexpected low caspase activity will occur. Turbidity, lipemia or particulate materials in samples can decrease the assay precision. * Also available as 50 Test size. Please inquire for catalog number and pricing. Product Catalog # Size Caspase-2 Colorimetric Assay* KHZ Tests Caspase-3 Fluorometric Assay* KHZ Tests Caspase-3 Colorimetric Assay* KHZ Tests Caspase-6 Fluorometric Assay* KHZ Tests Caspase-6 Colorimetric Assay* KHZ Tests Caspase-8 Fluorometric Assay* KHZ Tests Caspase-8 Colorimetric Assay* KHZ Tests Caspase-9 Colorimetric Assay* KHZ Tests Caspases -2, -3, -6, -8, -9 Colorimetric Assay Sampler Kit KHZ Tests Anti-caspase-1/ICE (human, mouse, rat) AHZ µg Anti-caspase-3 (human, mouse, rat) AHZ µg Anti-caspase-6 (human) AHZ µg Anti-caspase-8 (human), (Clone 5F7) AHZ µg Anti-caspase-8 (human), (Clone 12F5) AHZ µg Anti-caspase-10 (human) AHZ µg Recombinant Human Caspase-3/CCP32 PHZ Units Recombinant Human Caspase-6 PHZ Units

11 Chapter 3:PARP Introduction Protein cleavage, or proteolysis, is a general process in the cells and is involved in many cell activities including the activation of the pro-enzymes, production of functional proteins, and cell signaling. For example, under normal conditions, caspases are present in cells as inactive proenzymes, or procaspases. Upon induction of apoptosis, procaspases are activated through proteolytic processing. Active caspases lead to a self-amplifying cascade of proteolysis and cleavage of many cellular proteins including PARP (Poly (ADP- Ribose) Polymerase). PARP is a 116 kda nuclear protein which is strongly activated by DNA strand breaks. PARP plays a role in DNA repair as well as in other cellular processes, including DNA replication, cell proliferation and differentiation. During apoptosis, ICE family members, such as caspase-3 and -7, cleave PARP to yield an 85 kda and a 25 kda fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. BioSource International, Inc. offers an anti-parp-fitc conjugated Cleavage Site-Specific Antibody (CSSA) (Cat. #44-699) that can detect apoptotic cells by flow cytometry. An alternative to the TUNEL assay, the PARP-FITC CSSA can detect apoptosis in adherent and suspension cells. The PARP-FITC CSSA is also available in kit form (Cat. #AHM2011). An unconjugated PARP CSSA (Cat. #44-698) and a Biotin conjugated form (Cat. #44-697) can be used for other applications. 2% 52% 34% 22% Staurosporine dose-response HeLa cells. From left to right, untreated, 1.0, 0.5 and 0.25 µm staurosporine. M2 represents the percentage of apoptotic cells in each sample. Protocol for Staining Suspension Cells by Flow Cytometry 1. Treat Jurkat cells with 0.25, and 0.06 µg/ml of anti-fas mab (Cat.# AHS9552) for 1 hour. Use untreated Jurkat cells as a control. 2. Collect cells and wash 2 x in PBS. 3. Fix cells in IC Fix buffer (Cat. # FB001) containing 4% paraformaldehyde for 20 minutes at 4 C. 4. Permeablize cells in IC Perm buffer (Cat. # PB001) for 10 minutes. 5. Add 10 µl PARP-FITC CSSA (Cat. # ) to 10 5 cells in 100 µl IC Perm buffer and incubate for 30 minutes at RT. 6. Wash the cells 2 x in IC Perm buffer. 7. Resuspend cells in PBS and analyze cells by FACS. Detection Flow Cytometry IHC WB Methods Adherent Suspension PARP CSSA TUNEL Annexin-V +/ Protocol for Staining Adherent Cells by Flow Cytometry 1. Treat HeLa cells with 1, 0.5 and 0.25 µm staurosporine for 5 hours. Use untreated HeLa as a control. 2. Keep culture supernatant (some apoptotic cells are detached). 3. Wash cells 2 x in situ with PBS. 4. Add 2 mm EDTA to the cells and check under a microscope. 5. Once detached, collect cells and add to culture supernatant. 6. Wash cells 1 x with PBS. 7. Fix cells in IC Fix buffer (Cat. # FB001) containing 4% paraformaldehyde for 20 minutes at 4 C. 8. Permeablize cells in IC Perm buffer (Cat.# PB001) for 10 minutes. 9. Add 10 µl PARP-FITC CSSA (Cat. # ) to 10 5 cells in 100 µl IC Perm buffer and incubate for 30 minutes at RT. 10. Wash cells in IC Perm buffer. 11. Resuspend cells in PBS and analyze cells by FACS. Induction of apoptosis in Jurkat cells by anti-fas mab. From left to right, untreated, 0.25, 0.125, and 0.06 µg/ml anti-fas mab. M2 represents the percentage of apoptotic cells in each sample. Protocol for Immunoblotting 1. Perform SDS-PAGE on cell lysates treated with apopotsis inducers, or untreated cells (as a control) and transfer the protein onto PVDF membrane. 2. Block the membrane in Blocking Buffer (5% non-fat milk in ttbs) for 30 minutes at RT. 3. Incubate the membrane in 0.5 µg/ml of anti-parp CSSA (Cat. # ) in Blocking Buffer for 1 hour at RT. 4. Wash the membrane 3 x with Washing Buffer (ttbs). 5. Incubate the membrane with goat F(ab ) 2 anti-rabbit IgG alkaline phosphatase (Cat.# ALI4405) at 1:5000 dilution for 1 hour at RT. 6. Wash the membrane 3 x with Washing Buffer. 7. Develop in Tropix WesternStar buffer for 5 minutes and expose to film.

12 Chapter 4: BID Detection of PARP cleavage in HeLa cells by Western blotting. Protocol for Immunocytochemistry 1. Grow HeLa cells on chamber slide and treat with staurosporine. Use untreated cells as a control. 2. Discard culture supernatant and fix cells in cold acetone for 2 minutes. 3. Wash cells in PBS. 4. Block endogenous peroxidase by incubating cells on slides with 0.03 M NaN 3 and 0.03% H 2 O 2 in PBS for 10 minutes and rinse slide 3 x in PBS. 5. Block with Blocking Buffer (5% BSA in PBS) for 30 minutes at RT. 6. Apply anti-parp CSSA (cat. # ) at 0.5 µg/ml in Blocking Buffer to slide and incubate it for 1 hour at RT and rinse slide 3 x in PBS. 7. Apply biotinylated goat F(ab ) 2 anti-rabbit IgG (Cat. # ALI4409) at 1: 200 dilution in Blocking Buffer for 1 hour at RT and rinse slide 3 x in PBS. 8. Apply ABC reagent (Vectastain Elite) for 30 minutes and rinse slide 3 x in PBS. 9. Add DAB substrate for 5 minutes and wash the slide 3 x in dh 2 O. 10. Counterstain, dehydrate, and seal the slide with coverslip. BID,known as BH3 interacting domain death agonist, is an intracellular amplifier of apoptotic signals and is a BH3 domain only protein of the Bcl-2 family. Cleavage of BID plays an important role in the transduction and amplification of apoptotic signals during apoptosis. Typically, there are two major known apoptotic signal pathways during apoptosis: 1) The caspase-8 pathway initiated by stimulation of Fas or TNF-α (death receptor agonists) and 2) The Caspase-9/cytochrome c pathway induced by irradiation or cytotoxic drugs. Cleaved BID serves as a direct link between the caspase-8 pathway and caspase- 9/cytochrome c death machinery. BID usually exists as a 22 kda inactive protein in the cytosol of living cells. It becomes cleaved and activated by caspase-8 in response to apoptotic stimuli such as TNF-α, Fas and TRAIL. The cleavage of BID by caspase-8 occurs at the amino acid residues Asp59/Gly60 in mouse and Asp60/Gly61 in human. The C-terminal part of cleaved BID [p15] translocates onto mitochondria and triggers cytochrome c release. BioSource has 4 BID antibodies as part of our apoptosis product line. Two of these antibodies recognize human BID; one recognizes only full length human BID, the other recognizes the p15 portion of BID as well as full-length human BID (see figure below for data). The other two antibodies specifically recognize mouse BID; one recognizes only full length mouse BID, the other is a cleavage site-specific antibody (CSSA) to mouse BID [59/60]. The CSSA to mouse BID [59/60] specifically recognizes the 15 kda cleaved BID fragment. Staurosporine treated HeLa cells Full length BID Untreated Cleaved BID, p PARP CSSA stained apoptotic HeLa cells treated with staurosporine. Product Catalog # Size PARP [214/215] FITC Conjugated Kit AHM tests PARP [214/215] FITC CSSA tests PARP [214/215] CSSA Unconjugated µg PARP [214/215] CSSA Biotin tests Proteins were resolved from recombinant human BID (Lane 1), caspase-8 treated human BID (Lane 2), Jurkat cells (Lane 3), and the apoptotic Jurkat cells induced by anti-fas antibody (Cat. #AHS9552) (Lane 4) using SDS-PAGE. The proteins were incubated with the anti-human BID (p15) antibody at 0.5 µg/ml. The data show that the anti-human BID (p15) antibody recognizes the 15 kda BID fragment cleaved by caspase-8 and in apoptotic Jurkat cells (Lane 2 and 4).

13 Chapter 5: DNA Fragmentation Mouse L929 cells were treated with 5 µg /ml actinomycin plus 2.5 ng/ml, 5 ng/ml and 10 ng/ml mouse TNF-alpha (Cat. # PMC3014) for 5 hours (Lane 3, 4 and 5). Untreated L929 cells (Lane 1) and actinomycin treated L929 cells (Lane 2) were used as controls. Cells were incubated with the anti-mouse full length BID antibody (Cat. #44-434, Left panel) and anti-mouse BID CSSA [59/60] (Cat. #44-436, Right panel). The data show that anti mouse full length BID antibody only reacts with a 22 kda mouse full length BID (Left panel). The mouse BID CSSA [59/60] recognizes the cleaved mouse BID in apoptotic L929 cells (Right panel). Product Catalog # Size Anti-human BID [p15] mg Anti-human Full Length BID mg Anti-mouse BID [59/60] mg Anti-mouse Full Length BID mg Full length BID Cleaved BID Purpose: The ApoTarget Quick Apoptotic DNA Ladder Detection Kit is designed for preparation of nucleic acids from mammalian cells to determine the level of DNA fragmentation of apoptotic cells. Principle of the Method Internucleosomal DNA fragmentation is considered a hallmark of apoptosis. During apoptosis, activated nucleases degrade the higher order chromatin structure of DNA into fragments of 50 to 300 kilobases and subsequently into small DNA pieces of about 200 base pairs in length. These DNA fragments can be extracted from cells and visualized by horizontal gel electrophoresis followed by ethidium bromide staining. The detection of DNA fragments by gel electrophoresis is one method to identify cells undergoing apoptosis. The ApoTarget Quick Apoptotic DNA Ladder Detection Kit (Catalog #KHO1021) provides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other methods which require 1 to 2 days to finish, this detection method only requires less than 90 minutes to prepare DNA in a single tube, without the need for extractions or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis. This procedure increases recovery of small fragmented DNA and, therefore, improves the sensitivity of the assay. Reagents and Materials 1. TE Lysis Buffer 2. Enzyme A Solution 3. Enzyme B Solution 4. Ammonium Acetate Solution 5. DNA Suspension Buffer 6. Agarose and TBE Buffer (1 L TBE buffer contains 5.4 g Tris, 2.8 g Boric Acid, 2 ml of 0.5 M EDTA solution, ph 8.0) 7. PBS 8. Ethidium Bromide 9. Ethanol 10. DNA ladder marker 11. Microcentrifuge 12. DNA electrophoresis equipment 13. UV light source and camera Assay Procedure 1. Induce apoptosis in cells by desired method. Concurrently incubate a control without induction. Pellet x 10 6 cells in a 1.5 ml microcentrifuge tube. 2. When the induction is complete, wash cells with PBS and pellet cells by centrifugation for 5 minutes at 500 x g. 3. Carefully discard the supernatant. 4. Lyse the cells with 20 µl TE Lysis Buffer by carefully pipetting up and down several times.

14 5. Add 5 µl Enzyme A Solution to the crude lysate. Mix by gentle vortexing and incubate at 37 C in a waterbath for 10 minutes. 6. Add 5 µl Enzyme B Solution to each sample and incubate at 37 C for 30 minutes in a waterbath or until the lysate becomes clear. 7. Add 5 µl Ammonium Acetate Solution and 100 µl of absolute ethanol (kept at-20 C) to each sample. Vortex and allow the DNA to precipitate at -20 C for 10 to 15 minutes. 8. Centrifuge the sample for 10 minutes at 12,000 to 14,000 x g to collect the precipitated DNA. 9. Carefully discard the supernatant. 10. Add 0.5 ml of 70% cold ethanol to wash the DNA pellet and re-centrifuge the sample 10 minutes at 12,000 to 14,000 x g. 11. Discard the supernatant and air-dry the DNA pellet for 10 minutes at room temperature. 12. Add 30 µl of DNA Suspension Buffer, and resuspend the DNA by carefully pipetting up and down several times. 13. Load 15 to 30 µl of each sample onto a 1% agarose gel containing 0.5 µg/ml ethidium bromide in both gel and running buffer (1x TBE). 14. Run the gel at 5 V/cm for 1 to 2 hours. 15. Ethidium bromide-stained DNA can be visualized by transillumination with UV light and photographed. Typical Data Using DNA Ladder Detection Kit Figure 1: 1 2 Apoptosis was induced in Jurkat cells by incubating cells with 2 µm Camptothecin for 6 hours at 37 C (Lane 2). Jurkat cells without Camptothecin were used as control (Lane 1). Detection of DNA fragmentation was performed as described above. Product Catalog # Size Quick Apoptotic DNA Laddering Kit KHO tests Chapter 6: TUNEL Detection Introduction of TUNEL Assay Cell death by apoptosis is characterized by DNA fragmentation in and/or kilobases. Further internucleosomal DNA fragmentation in base pairs may also occur. Such characteristics have been used to distinguish apoptotic cells from normal or necrotic cells. To detect apoptotic cells, whatever the pattern of DNA fragmentation, the TUNEL (Terminal deoxynucleotidyl transferase (TdT) mediated dutp Nick End Labeling) method is commonly utilized. In the TUNEL assay (Apo-BrDU) exogenous TdT is used to catalyze a template-independent addition of bromodeoxyuridine triphosphates (Br-dUTP) to the free 3 -hydroxyl ends of double or single stranded DNA fragments. After incorporation, the labeled BrDU can be identified by FITC conjugated anti-bromodeoxyuridine (BrDU) antibodies and analyzed using a flow cytometer or a fluorescence microscope. Due to the many free 3 -hydroxyl ends of fragmented DNA in apoptotic cells, a good signal is generated in affected cell populations. These cells can be visualized in tissue sections or quantified with flow cytometry. Compared to a single-step labeling FITC conjugated dutp, this two step method provides a more sensitive, stronger signal. Method: ApoBrDU Detection Description of Kit The BioSource International, Inc. APO-BRDU Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells including positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl tranferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), fluorescein labeled anti-brdu antibody for labeling DNA breaks and propidium iodide/rnase A solution for counterstaining the total DNA. Reagents and Materials 1. Positive Control Cells 2. Negative Control Cells 3. Wash Buffer 4. Reaction Buffer 5. TdT Enzyme 6. Br-dUTP 7. Rinsing Buffer 8. Fluorescein~PRB-1 mab 9. I/RNase Staining Buffer 10. Flow Cytometer 11. Distilled water 12. 1% (w/v) paraformaldehyde (methanol free) in Phosphate Buffered Saline (PBS) % (v/v) ethanol

15 C water bath 15. Ice bucket x 75 mm flow cytometry test tubes 17. Pipettes and pipetting aids Summary Diagram of APO-BRDU Methodology Measurable Features of Apoptosis One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells which remains largely intact does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3 -hydroxyl ends in the DNA. This property can be used to identify apoptotic cells by labeling the 3 -hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of deoxyribonucleoside triphosphates to the 3 -hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-BRDU Kit. Recent evidence has demonstrated that Br-dUTP is more readily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin. This greater incorporation gives rise to a stronger flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled anti-brdu monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP owing to the lack of exposed 3 - hydroxyl DNA ends. Induce apoptosis in cells Fix cells (see Page 24) Wash cells Label DNA in cells Rinse cells Fixed control cells from kit Stain cells with Fluorescein~PRB-1 TdT + Br-dUTP Fluorescein PRB-1 Propidium Iodidie RNAse A Treatment DNA Strand Breaks Add Br-dUTP To 3 -OH DNA Ends Antibody Labeled Break Sites Figure 1: Diagrammatic representation of the addition of bromodeoxyuridine triphosphate (Br-dUTP) catalyzed by terminal deoxynucleotidyl transferase (TdT) to the 3 -OH sites of DNA strand breaks induced in the genome of apoptotic cells. Flow Cytometry analysis Figure 2: Flow diagram used in the APO-BRDU Apoptosis Assay. The positive and negative control cells are supplied in the kit and are already fixed. The cells supplied by the researcher should be fixed by the researcher according to a protocol suggested on next page.

16 Cell Fixation Procedure for APO-BRDU Assay NOTE: Cell fixation using paraformaldehyde is a required step in the APO-BRDU assay. The following cell fixation procedure is a suggested method. Variables such as cell origin and growth conditions can affect the results. The fixation conditions provided below should be considered as guidelines. Additional experimentation may be required to obtain results comparable to the control cells provided with the kit. The positive and negative control cells provided in the APO-BRDU KIT are already fixed. 1. Suspend 1-2 x 10 6 cells in 0.5 ml of 10 mm sodium phosphate ph 7.2, 150 mm sodium chloride (PBS). 2. Add the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes. 3. Centrifuge cells for 5 minutes at 300 x g and discard the supernatant. 4. Wash the cells in 5 ml of PBS then pellet the cells by centrifugation. Repeat the wash and centrifugation. 5. Resuspend the cells in 0.5 ml of PBS. 6. Add cells to 5 ml of ice-cold 70% (v/v) ethanol. Let cells stand for a minimum of 30 minutes in ice or the freezer. See note below. 7. Store cells in 70% (v/v) ethanol at -20 C until use. Cells can be stored at -20 C several days before use. Note: In some biological systems storage of the cells at -20 C in 70% (v/v) ethanol for at least hours prior to staining for apoptosis detection yields the best results. APO-BRDU PROTOCOL The following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the APO-BRDU kit. The same procedure should be employed for measuring apoptosis in the cell specimens provided by the researcher. 1. Resuspend the positive and negative control cells by swirling the vials. Remove 1 ml aliquots of the control cell suspensions (approximately 1 x 10 6 cells per 1 ml) and place in 12 x 75 mm flow cytometry centrifuge tubes. Centrifuge (300 x g) the control cell suspensions for 5 minutes and remove the 70% (v/v) ethanol by aspiration, being careful to not disturb the cell pellet. 2. Resuspend each tube of control cells with 1 ml of Wash Buffer for each tube. Centrifuge as before and remove the supernatant by aspiration. 3. Repeat the Wash Buffer treatment (step 2). 4. Resuspend each tube of the control cell pellets in 50 µl of the DNA Labeling Solution (prepared as described below). DNA Labeling Solution 1 Assay 5 Assays 10 Assays TdT Reaction Buffer 10 µl 50 µl 100 µl TdT Enzyme 0.75 µl 3.75 µl 7.5 µl Br-dUTP 8 µl 40 µl 80 µl Distilled H 2 O µl µl µl Total Volume 51 µl 255 µl 510 µl The appropriate volume of Staining Solution to prepare for a variable number of assays is based upon multiples of the component volumes combined for 1 assay. Mix only enough DNA Labeling Solution to complete the number of assays prepared per session. The DNA Labeling Solution is active for approximately 24 hours. 5. Incubate the cells in the DNA Labeling Solution for 60 minutes at 37 C in a temperature controlled bath. Shake cells every 15 minutes to resuspend. NOTE: The DNA Labeling Reaction can also be carried out at C overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37 C may need to be adjusted to longer or shorter periods depending on the characteristics of the cells supplied by the researcher. 6. At the end of the incubation time, add 1.0 ml of Rinse Buffer to each tube and centrifuge each tube (300 x g) for five minutes. Remove the supernatant by aspiration.

17 7. Repeat the cell rinsing (as in step 6) with 1.0 ml of the Rinse Buffer (red cap), centrifuge and remove the supernatant by aspiration. 8. Resuspend the cell pellets in 0.1 ml of the Antibody Solution (prepared as described below). Antibody Solution 1 Assay 5 Assays 10 Assays Fluorescein~PRB-1 (orange cap) 5 µl 25 µl 50 µl Rinse Buffer (red cap) 95 µl 475 µl 950 µl Total Volume 100 µl 500 µl 1000 µl 9. Incubate the cells with the Fluorescein~PRB-1 Antibody Solution in the dark for 30 minutes at room temperature. Hint: Wrap tubes with aluminum foil. 10. Add 0.5 ml of the Propidium Iodide/RNase A Solution to the tube containing the 0.1 ml Antibody Staining Solution. Note: If the cell density is low, decrease the amount of PI/RNase A solution to 0.3 ml. 11. Incubate the cells in the dark for 30 minutes at room temperature. Analyzing the APO-BRDU Samples on the Flow Cytometer This assay is run on a flow cytometer equipped with a 488 nm Argon laser as the light source. Propidium Iodide (total cellular DNA) and Fluorescein (Apoptotic Cells) are the two dyes being used. Propidium Iodide (PI) fluoresces at about 623 nm and Fluorescein at 520 nm when excited at 488 nm. No fluorescence compensation is required. Two dual parameter and two single parameter displays are created with the flow cytometer data acquisition software. The gating display should be the standard dual parameter DNA doublet discrimination display with the DNA Area signal on the Y-axis and the DNA Width on the X-axis (Becton-Dickinson, see Figure 4) or DNA Peak/Integral signal for Coulter, see Figure 5 on page 29. From this display, a gate is drawn around the nonclumped cells and the second gated dual parameter display is generated. The normal convention of this display is to put DNA (Linear Red Fluorescence) on the X-axis and the FITC~PRB-1 (Log Green Fluorescence) on the Y-axis (see bottom display next page). Two single parameter gated histograms, DNA and FITC~PRB-1, can also be added but are not necessary. By using the dual parameter display method, not only are apoptotic cells resolved, but at which stage of the cell cycle they are in is also determined. The Log Green Fluorescence histograms of the control cells should look like Figure 3 below. 12. Analyze the cells in Propidium Iodide/RNase Solution by flow cytometry. Negative Control Cells Positive Control Cells 13. Analyze the cells within 3 hours of staining. Relative Cell Number Relative Cell Number Apoptotic Cells Log Green Fluorescence Figure 3: Flow Cytometry Data of APO-BRDU Negative & Positive Control Cells.

18 Flow Cytometer Setup for Becton Dickinson Hardware Display 1 Flow Cytometer Setup for Coulter Hardware Display 1 Technical Tips and Frequently Asked Questions About the APO-BRDU Assay DNA Area DNA Width Gate DNA Peak DNA Int. Gate 1. For those researchers using adherent cell line systems, the cells in the supernatant have a higher probability of being apoptotic than do the adherent cells. Save cells in the supernatant for assay prior to trypsinization of the adherent cell layer. 2. Cell fixation using a DNA cross-linking chemical fixative is an important step in analyzing apoptosis. Unfixed cells may lose smaller fragments of DNA that are not chemically fixed in place inside the cell during washing steps. The researcher may have to explore alternative fixation and permeabilization methods to fully exploit their systems. FITC ~PBR-1 Gated From Display 1 DNA Area Figure 4: APO-BRDU TM Positive Control Cells Positive Apoptotic Cells Non-Apoptotic Cells Typical FACScan TM Gain Settings Parameter Amplifier Gain Detector Gain FL 1 Log 380 Volts FL Volts FL 3 Width.87 FL 3 Area 3.25 Threshold- FL 3, 40 FITC ~PBR-1 Gated From Display 1 DNA Int. Figure 5: APO-BRDU TM Positive Control Cells Positive Apoptotic Cells Non-Apoptotic Cells Typical XL TM Gain Settings Parameter Amplifier Gain Detector Gain FL 1 Log 589 Volts FL Volts AUX(FL3 Peak) Volts Discriminator-AUX (FL3 Peak) 3. A cytospin or centrifugal cytology slide can be prepared from APO-BRDU samples in the following manner. After completion of the Fluorescein~PRB-1 antibody staining, but prior to the Propidium Iodide/RNAse A treatment, put a drop of the stained cells on a slide, spin it and observe the sample under a fluorescence microscope. 4. Surface marker staining of cellular antigens can be accomplished by first incubating the cells with the fluorescent-labeled CD antibody, followed by fixing and permeabilizing the cells prior to testing in the APO-BRDU Assay. 5. To minimize cell loss during the assay, restrict the assay to the use of a single 12 x 75 mm test tube. If polystyrene plastic test tubes are used, an electrostatic charge can build up on the sides of the tube. Cells will adhere to the side of the tube and the sequential use of multiple tubes can result in significant cell loss during the assay. 6. Occasionally a mirror image population of cells at lower intensity is observed in the flow cytometry dual parameter display. This population arises because during the 50 µl DNA Labeling Reaction, some cells have become stuck to the side of the test tube and are not fully exposed to the reaction solution. This phenomenon can be overcome by washing all the cells from the side of the tube and making sure all cells are properly suspended at the beginning of the labeling reaction. 7. If a low intensity of fluorescein staining is observed, try increasing the incubation time during the 50 µl DNA Labeling Reaction. Some researchers have found labeling times of up to four hours at 37 C may be required for certain cell systems. 8. If the DNA cell cycle information is not required, it is not necessary to add the PI/RNase A solution to each tube. Product Catalog # Size APO-BRDU Assay (TUNEL) KHO Tests