Flow Cytometry Cell Analysis with Molecular Probes and Caltag Andrea Hamm-Baarke, TSS for Cell Separation and Analys

2. 2 Agenda Today, the state of our industry is strong, and getter stronger. Let me share some data with you to underline this point. First, important to many of you in this room, our financial situation is greatly improved. Biotech financing is steadily improving, with more than $14B raised so far this year, compared to $12B in the first 9 months of 2003, and $9 B in all of 2001 There is no doubt that Imclone and others have caused investors to put a premium on lower risk, later stage developments, but companies at various stages are still attracting capital. We also see much greater stability � in terms of cash on hand � with nearly 70% of companies in the industry holding more than two years� cash. Today, the state of our industry is strong, and getter stronger. Let me share some data with you to underline this point. First, important to many of you in this room, our financial situation is greatly improved. Biotech financing is steadily improving, with more than $14B raised so far this year, compared to $12B in the first 9 months of 2003, and $9 B in all of 2001 There is no doubt that Imclone and others have caused investors to put a premium on lower risk, later stage developments, but companies at various stages are still attracting capital. We also see much greater stability � in terms of cash on hand � with nearly 70% of companies in the industry holding more than two years� cash.

3. 3 Cellular Immunology

5. 5 Introduction to Flow Cytometry Flow Cytometry Is a commonly used tool to analyze and/or isolate single cell suspensions. The cells are typically stained with light fluorescent dyes, placed in a fluid stream and pass as single cell a Laser. The emitted light measures several parameters like: Size of the cell Granularity of the cell Emitted light of dyes (if the cells are stained) Flow Cytometry is used in all branches of Medicine e.g. : Diagnostic (e.g. CD4 to CD8 Ratio in HIV Patients) Oncology (specific cancer markers, apoptosis) Immunology (phenotype analysis) Dermatology (Allergy)

6. 6 Introduction to Flow Cytometry

7. 7 FACS= Fluorescence Activated Cell Sorter

8. 8 Flow Cytometry

9. 9 Fluorescence Dye Labeled Antibodies

10. 10 The optimal Fluorochrom is:

11. 11 Analysis Datas: Dot Blot

12. 12 Fluorescence Dyes

13. 13 AF647 = Cy5. AF633 = APC?AF647 = Cy5. AF633 = APC?

14. 14 Products CD Antigens, such as CD3, CD4, CD8 Non-CD Antigens, such as HLA-DR, ZAP-70, Fetal Hemoglobin Pre-mixed cocktails (mostly for clinical labs) Immunoglobulins, such as IgG, IgM, Kappa, Lambda CD Antigens, such as CD3, CD4, CD8 Non-CD Antigens, such as F4/80, Gr-1, NK1.1 Immunoglobulins, such as IgG, IgM, Kappa, Lambda MHC Antigens (Class I and Class II) T-cell Receptor Antigens, such as KJ1-26, TCR V alpha 2

15. 15 Products Antibodies to Rat Antigens CD Antigens, such as CD3, CD4, CD8 Non-CD Antigens, such as Pan B, NKR-P1a Immunoglobulins, such as IgG, IgM, Kappa, Lambda Antibodies to Cytokines and Chemokines Human, such as Interferon gamma, IL-2, Rantes, CXCR4 Mouse, such as IL-2, Interferon gamma Isotype Controls (�Negative� controls) Singles Pre-mixed Sample Preparation Lysing Reagents Fix & PermTM Caltag Counting Beads

16. 16 Flow Cytometry

17. 17

18. 18 These graphs just show how fast the complex formation is. Firstly, we recommend 5 minutes for your antibody incubation step however you can see that complex formation is in actual fact a lot faster. Secondly, complex dissassociation is very slow. These graphs just show how fast the complex formation is. Firstly, we recommend 5 minutes for your antibody incubation step however you can see that complex formation is in actual fact a lot faster. Secondly, complex dissassociation is very slow.

19. 19 Why use ZENON? Simple No need to use secondary antibodies anymore Speed Zenon labeling complexes are ready to use for cell staining within 5 minutes� Quantitative Labeling 100% of the primary antibody sample is labeled. No Preparation Removal of exogenous proteins such as serum albumin from primary antibody samples is unnecessary. Compatibility Multiple Zenon One�labeled mouse antibodies can be used in the same immunolabeling protocol. Economy A standard Zenon labeling requires only 1 �g of primary antibody (versus 100 �g for chemical labeling). Save time and money.

20. 20 Analysis of the cell status

21. 21 Flow Cytometry

22. 22 Cell Cycle Analysis Cellular events and nuclear events in flow cytometry often describe the distribution of a population of cells in the different nuclear phases.Cellular events and nuclear events in flow cytometry often describe the distribution of a population of cells in the different nuclear phases.

23. 23 Vybrant� DyeCycle� Orange Stain Use linear fluorescenceUse linear fluorescence

24. 24 Permeant Nucleic Acid Dyes Dyes which have the ability to penetrate an intact cell membrane to stain nucleic acid are classified as live-cell, or cell-permeant nucleic acid dyes. These dyes can be used for determining the DNA content of viable cells. Examples include: Hoechst dyes dsDNA Vybrant � DyeCycle� Violet stain dsDNA Vybrant � DyeCycle� Green stain dsDNA Vybrant � DyeCycle� Orange stain dsDNA SYTO� 13 stain DNA/RNA Hoechst dyes (UV excitation) and DyeCycleViolet bind in the minor groove region of DNA with preference for AT groups. DyeCycle Green and DyeCycle Orange probably are intercalating dyes. SYTO dyes not DNA specific, but the DyeCycle stains and Hoechst dyes ARE specific for DNA. With DNA-selective dyes, Cells are incubated in the presence of dye and NO additional treatment is necessary (ie fix/perm/RNase), which allows for the possibility of sorting Hoechst dyes (UV excitation) and DyeCycleViolet bind in the minor groove region of DNA with preference for AT groups. DyeCycle Green and DyeCycle Orange probably are intercalating dyes. SYTO dyes not DNA specific, but the DyeCycle stains and Hoechst dyes ARE specific for DNA. With DNA-selective dyes, Cells are incubated in the presence of dye and NO additional treatment is necessary (ie fix/perm/RNase), which allows for the possibility of sorting

25. 25 Flow Cytometry

26. 26 Viability: Cell membrane forms intact barrier Vitality: Cell mediates active processes Viability & Vitality Viability and vitality are principally used to enumerate the proportion of live and dead cells in a population. The diversity of live cells and their environments makes it impossible to devise a single assay applicable to all cell types. Viability is often used to refer to the integrity of the plasma membrane. Cells that successfully exclude impermeant reagents, such as trypan blue or propidium iodide, are considered viable. Vitality is often used to refer to cells that display active metabolic or energetic processes. Cells containing esterases that cleave non-fluorescent carboxyfluorescein analogs to fluorescent fluorescein are considered vital. There is obviously a gray zone between these determinations: cells may show enzyme activity after the plasma membrane starts to degrade; cells may lose some metabolic functions before membrane damage becomes apparent. Viability and vitality are principally used to enumerate the proportion of live and dead cells in a population. The diversity of live cells and their environments makes it impossible to devise a single assay applicable to all cell types. Viability is often used to refer to the integrity of the plasma membrane. Cells that successfully exclude impermeant reagents, such as trypan blue or propidium iodide, are considered viable. Vitality is often used to refer to cells that display active metabolic or energetic processes. Cells containing esterases that cleave non-fluorescent carboxyfluorescein analogs to fluorescent fluorescein are considered vital. There is obviously a gray zone between these determinations: cells may show enzyme activity after the plasma membrane starts to degrade; cells may lose some metabolic functions before membrane damage becomes apparent.

27. 27 Viability & Vitality: LIVE/DEAD� Kits Molecular Probes provides a number LIVE/DEAD� kit combinations that can provide viability or vitality assessments for a variety of experimental conditions. LIVE/DEAD Viability Assay kits provide the reagents with simple protocols for fluorescence-based assessment of numbers of live and dead cells. LIVE/DEAD Viability / Cytotoxicity Kit. Calcein AM and ethidium homodimer-1 provide an accurate general-purpose assay of cytotoxic events.1-3 Calcein AM is cleaved by cell esterases to form green-fluorescent calcein; ethidium homodimer-1 is an impermeant dye that fluoresces red in the nuclei of dead cells. This assay is more dependable than those using trypan blue, and it is faster, less expensive and safer than 51Cr-release assays. The ease, reliability and low cost of this kit make it economical for high-throughput screening of cytotoxic agents. LIVE/DEAD Cell-Mediated Cytotoxicity Kit. The two-color fluorescence assay results correlate well with those obtained using the conventional 51Cr-release cell-mediated cytotoxicity assay.4 DiOC18(3) is a membrane probe that labels all cells; propidium iodide labels dead cells. Samples may be monitored for cell-mediated toxicity over the course of many hours without the hazards and disposal problems encountered using radioactive probes. LIVE/DEAD Reduced Biohazard Viability / Cytotoxicity Kits. The viability stains used in these kits remain in place after the sample is fixed (allows inactivation of pathogens). Samples may be fixed at various times during an experiment and analyzed up to several hours later. Kits #2-#4 (L23101, L23102, L23105) only contain one fluorescent dye and are based on the principle of cytoplasmic exclusion in viable cells. Basically, Live cells only react weakly to the fluorescent molecules on the cell surface. Dead cells with compromised plasma membranes allow the fluorescent dye to enter the cytoplasm generating a significantly brighter signal. LIVE/DEAD Sperm Viability Kit. SYBR� 14 dye stains the DNA of all cells; propidium iodide stains the DNA of damaged cells. Assessing sperm viability with this assay is accomplished in only 5-10 minutes, much more rapidly than with conventional stains. The fluorescent stains in the kit are excited in the visible wavelength range, avoiding harm to the sperm from exposure to UV. Researchers have documented the use of this kit with a variety of species and experimental conditions.5-9 This kit can be adapted to other cell types. 1. J Immunol Methods 222, 31 (1999); 2. Photochem Photobiol 63, 111 (1996); 3. J Neurosci 15, 5389 (1995); 4. J Immunol Methods 222, 31 (1999); 5. Theriogenology 45, 923 (1996); 6. Biol Reprod 56, 991 (1997); 7. J Androl 18, 324 (1997); 8. Mol Reprod Devel 46, 408 (1997); 9. Reprod Fertil Devel 8, 1165 (1996) Molecular Probes provides a number LIVE/DEAD� kit combinations that can provide viability or vitality assessments for a variety of experimental conditions. LIVE/DEAD Viability Assay kits provide the reagents with simple protocols for fluorescence-based assessment of numbers of live and dead cells. LIVE/DEAD Viability / Cytotoxicity Kit. Calcein AM and ethidium homodimer-1 provide an accurate general-purpose assay of cytotoxic events.1-3 Calcein AM is cleaved by cell esterases to form green-fluorescent calcein; ethidium homodimer-1 is an impermeant dye that fluoresces red in the nuclei of dead cells. This assay is more dependable than those using trypan blue, and it is faster, less expensive and safer than 51Cr-release assays. The ease, reliability and low cost of this kit make it economical for high-throughput screening of cytotoxic agents. LIVE/DEAD Cell-Mediated Cytotoxicity Kit. The two-color fluorescence assay results correlate well with those obtained using the conventional 51Cr-release cell-mediated cytotoxicity assay.4 DiOC18(3) is a membrane probe that labels all cells; propidium iodide labels dead cells. Samples may be monitored for cell-mediated toxicity over the course of many hours without the hazards and disposal problems encountered using radioactive probes. LIVE/DEAD Reduced Biohazard Viability / Cytotoxicity Kits. The viability stains used in these kits remain in place after the sample is fixed (allows inactivation of pathogens). Samples may be fixed at various times during an experiment and analyzed up to several hours later. Kits #2-#4 (L23101, L23102, L23105) only contain one fluorescent dye and are based on the principle of cytoplasmic exclusion in viable cells. Basically, Live cells only react weakly to the fluorescent molecules on the cell surface. Dead cells with compromised plasma membranes allow the fluorescent dye to enter the cytoplasm generating a significantly brighter signal. LIVE/DEAD Sperm Viability Kit. SYBR� 14 dye stains the DNA of all cells; propidium iodide stains the DNA of damaged cells. Assessing sperm viability with this assay is accomplished in only 5-10 minutes, much more rapidly than with conventional stains. The fluorescent stains in the kit are excited in the visible wavelength range, avoiding harm to the sperm from exposure to UV. Researchers have documented the use of this kit with a variety of species and experimental conditions.5-9 This kit can be adapted to other cell types. 1. J Immunol Methods 222, 31 (1999); 2. Photochem Photobiol 63, 111 (1996); 3. J Neurosci 15, 5389 (1995); 4. J Immunol Methods 222, 31 (1999); 5. Theriogenology 45, 923 (1996); 6. Biol Reprod 56, 991 (1997); 7. J Androl 18, 324 (1997); 8. Mol Reprod Devel 46, 408 (1997); 9. Reprod Fertil Devel 8, 1165 (1996)

28. 28 Loss of Membrane Asymmetry: Annexin V Loss of membrane asymmetry, characterized by an increased expression of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, is a hallmark of apoptosis. Annexin V is widely used to detect elevated expression of PS on the outer surface of the cell. Molecular Probes offers an extensive selection of fluorescent annexin V conjugates, shown above with their peak (excitation/emission) wavelengths. We have also bundled these into easy to use combinations with impermeant dyes and other reagents: the Vybrant line of kits. Loss of membrane asymmetry, characterized by an increased expression of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, is a hallmark of apoptosis. Annexin V is widely used to detect elevated expression of PS on the outer surface of the cell. Molecular Probes offers an extensive selection of fluorescent annexin V conjugates, shown above with their peak (excitation/emission) wavelengths. We have also bundled these into easy to use combinations with impermeant dyes and other reagents: the Vybrant line of kits.

29. 29 Loss of Membrane Asymmetry: Annexin V In viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. As cells undergo apoptosis, PS is translocated to the outer leaflet of the plasma membrane and exposed to the extracellular environment.1 The human vascular anti-coagulant, annexin V, is a 35-36 kD Ca2+-dependent phospholipid-binding protein that has a high affinity for PS.2 Annexin V, labeled with a fluorophore or biotin, can identify apoptotic cells by binding to PS exposed on the outer leaflet (see cartoon).3 Molecular Probes has collaborated with NeXins Research BV, the original developer of fluorescent PS-binding proteins, to provide the brightest annexin V conjugates available.These conjugates are included in many of the Vybrant Apoptosis Assay kits. Most of these kits contain labeled annexin V as well as a cell impermeant nucleic acid stain to serve as a label for late apoptotic and/or necrotic cells (see cartoon). A typical annexin V staining result is shown in the dot plots to the right. Jurkat cells, either untreated (top) or induced to apoptosis with camptothecin (bottom), were stained with Alexa Fluor 488 annexin V and Propidium iodide (Vybrant Apoptosis Assay Kit #2, V13241). The camptothecin-treated cells in the bottom plot show an elevated number of apoptotic and dead cells. 1. Cytometry 31, 1 (1998); 2. J Biol Chem 265, 4923 (1990); 3. Blood 84, 1415 (1994); In viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. As cells undergo apoptosis, PS is translocated to the outer leaflet of the plasma membrane and exposed to the extracellular environment.1 The human vascular anti-coagulant, annexin V, is a 35-36 kD Ca2+-dependent phospholipid-binding protein that has a high affinity for PS.2 Annexin V, labeled with a fluorophore or biotin, can identify apoptotic cells by binding to PS exposed on the outer leaflet (see cartoon).3 Molecular Probes has collaborated with NeXins Research BV, the original developer of fluorescent PS-binding proteins, to provide the brightest annexin V conjugates available.These conjugates are included in many of the Vybrant Apoptosis Assay kits. Most of these kits contain labeled annexin V as well as a cell impermeant nucleic acid stain to serve as a label for late apoptotic and/or necrotic cells (see cartoon). A typical annexin V staining result is shown in the dot plots to the right. Jurkat cells, either untreated (top) or induced to apoptosis with camptothecin (bottom), were stained with Alexa Fluor 488 annexin V and Propidium iodide (Vybrant Apoptosis Assay Kit #2, V13241). The camptothecin-treated cells in the bottom plot show an elevated number of apoptotic and dead cells. 1. Cytometry 31, 1 (1998); 2. J Biol Chem 265, 4923 (1990); 3. Blood 84, 1415 (1994);

30. 30 Selection Guide for Vybrant Apoptosis Assay Kits The table above shows the existing Vybrant kits and their primary features. The table above shows the existing Vybrant kits and their primary features.

31. 31 Online Resource for Information

32. 32 Where do we differentiate Widest range of fluorochromes available Key tandems Caltag Antibodies with Molecular Probes Dyes Custom conjugation program Alexa Dyes, Brighter and more Stabile Zenon Technology to label your own antibody First to market with key products Fetal hemoglobin ZAP-70 Pacific Blue, Pacific Orange antibody conjugate Cost-effective Huge Portfolio for Cell Analysis Cell Cycle Apoptosis Live/ Dead Cell Detection

33. 33 Flow Cytometry Resources

34. 34 Molecular Probes Handbook

35. 35 Catalogues