Applications of Cell Analysis Using Cytometric Approaches J.Paul Robinson SNP Professor of Cytomics February 14, 2006 This...
What can flow cytometry be used for? <ul><li>Immunology </li></ul><ul><li>Hematology </li></ul><ul><li>Pathology </li></ul...
CELLULAR ANTIGENS Adhesion Receptors Metabolic cytokines structure enzymes Slide courtesy of Jim Bender T cells B Cells Ph...
Cluster Designations  (CD) <ul><li>These are based on the Immunology Workshop an international committee that meets in Bos...
Immunofluorescence  staining specific binding nonspecific binding Slide from Dr. Carleton Stewart
Direct staining <ul><li>Fluorescent probe attached to antibody </li></ul><ul><li>Specific signal: weak, 3dyes/site </li></...
Avidin-Biotin method I biotinylated primary Ab biotin avidin biotinylated dye
CFU-GM MYELOBLAST MYELOCYTE META- MYELOCYTE BAND PMN CD16 CD11b CDw13 CD33 CD34 HLA-Dr  CD38  CD71  MY8 Myelomonocytic Ant...
From Duque et al, Clin.Immunol.News.  Negative Positive Decision Tree in Acute Leukemia HLA-DR T CD13,33 CD19 TdT CD10 CD2...
What are the principles in flow cytometry? <ul><li>Light   scatter ed by a laser or arc lamp </li></ul><ul><li>Specific  f...
Concepts <ul><ul><ul><li>Scatter :  Size, shape, granularity, polarized  scatter (birefringence), structure </li></ul></ul...
Clinical Analyzers
Cell Sorters (FACS – Fluorescence Activated Cell Sorter)
Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser  Flow cell PMT 3 Scatter Sensor Sample
Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter S...
 
 
Light Scatter <ul><li>Materials scatter light at wavelengths at which they do not absorb </li></ul><ul><li>If we consider ...
Optics for forward scatter scatter detector iris blocker Laser beam Stream in air or a round capillary
Frequency distribution histogram Number of events Intensity of parameter (e.g. fluorescence)
histogram Intensity of parameter Number of events
Flow cytometry measurements L M G SCATTER       FLUORESCENCE IMAGE
0 200 400 600 800 1000 Side Scatter Projection Light Scatter Gating Forward Scatter Projection 90 Degree Scatter Neutrophi...
Different size cells Particle or cell size (log scale) Number of events small large 0.1  1  10  100  1000 0.9 20 90 200 70...
Light Scatter of white blood cells <ul><li>Light scatter can be used to identify populations of cells </li></ul>x In perip...
Fluorescence - e.g. Monoclonal Antibodies
“ B” Cells “ T” Cells
3 Parameter Data Display Isometric Display
The Cell Cycle G 1 M G 2 S G 0 Quiescent cells
Definitions & Terms <ul><li>Ploidy </li></ul><ul><ul><li>related to the number of chromosomes in a cell </li></ul></ul><ul...
Normal Cell Cycle  G 0 G 0 - G 1 s DNA Content 2N 4N G 2 M G 0 G 1 s Cell Count G 2 M 0 200 400 600 800 1000 0 75 150 225 ...
A typical DNA Histogram G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events 2n 4n
Analyzing the DNA Histogram
Chromosome Analysis Most human chromosomes can be separated by flow cytometry
Chromosome Analysis (Bivariate   Flow Karyotyping - porcine) chromosome 1 chromosome 2
log Thiazole Orange .1 1000 100 10 1 Count 0 150 112 75 37 RMI  = 0 log Thiazole Orange .1 1000 100 10 1 Count 0 150 112 7...
4 colors - simultaneous collection (can go to 17 colors) Emission wavelength (nm) 530    580 630  680  730  780 FITC PE PE...
CD3 CD3 CD3 CD56 CD56 CD8 FOUR COLOR PATTERN CD4 CD8 CD56 - NK CD8 CD4 CD4 Data from Dr. Carleton Stewart CD56 – NK Cells ...
Multicolor Analysis Roederer, et al
Cellular  Response: <ul><li>Cell death </li></ul><ul><li>Cell ‘suicide’ </li></ul><ul><li>Ignore damage </li></ul><ul><li>...
Functional Assays <ul><li>intracellular pH </li></ul><ul><li>intracellular calcium </li></ul><ul><li>intracellular glutath...
TIME (seconds) 0 2400 1800 1200 600 Scale 345 115 38 12 4 Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Unstimulat...
FITC-Labeled Bacteria Phagocytosis
Cellular Functions <ul><li>Cell Viability </li></ul><ul><li>Phagocytosis </li></ul><ul><li>Organelle Function </li></ul><u...
Organelle Function <ul><li>Mitochondria Rhodamine 123 </li></ul><ul><li>Endosomes Ceramides </li></ul><ul><li>Golgi BODIPY...
Fluorescent Indicators <ul><li>How the assays work: </li></ul><ul><li>Superoxide :  Utilizes hydroethidine the sodium boro...
DCFH-DA  DCFH  DCF Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dich...
Phagosome O 2 O 2 - H 2 O 2 NADPH + H + NADP + HMP NADPH Oxidase GSSG GSH GR GP H 2 O 2 SOD O 2 - H + H 2 O Catalase H 2 O...
Cell Sorting <ul><li>Physically separating cells based on some measurable characteristic </li></ul><ul><li>Placing these c...
488 nm laser + - Fluorescence Activated  Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluor...
SMALL BEAD LARGE BEAD Frequency Histogram SMALL BEAD LARGE BEAD Sample in Sheath Sheath in Laser beam Stream  Charge +2KV ...
Cell Sorting Video of the  droplet formation in a sort stream from a Cytomation instrument.  Source: Purdue CDROM vol 4, 1...
Bindley Bioscience Center Discovery Park
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Large Animal Residents Lecture Feb 14, 2006

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  • In the most recent leukocyte typing workshop over 160 antibodies were classified into defined groups. These antibodies bind to cytokines or chemokine receptors, sensory molecules, cytokine or chemokine ligands, metabolic proteins, adhesion molecules, enzymes, structural proteins and other important functional proteins within the cell. These antibodies can be used to identify specific cell subsets by their unique repertoire of molecular expression as well as their functional state using multiparameter flow cytometry.
  • Hydrodynamic Systems This slide is similar to the previous slide except it shows how the sheath and sample are combined to make a laminar flow with the samples in the center of the stream.
  • 17
  • 12
  • use of maturity index decreases interlab variability RMI = fraction of highly fluorescent retics/total retics cursor is set to exclude nucleated cells
  • As additional antibodies are combined, further subsetting can be achieved and heretofore unknown populations of cells discovered (CD3+CD4+CD8-CD56+). The increased dimensionality of data may make its visualization difficult.
  • Large Animal Residents Lecture Feb 14, 2006

    1. 1. Applications of Cell Analysis Using Cytometric Approaches J.Paul Robinson SNP Professor of Cytomics February 14, 2006 This lecture can be found on http://www.cyto.purdue.edu/class NOTE: Many of the slides in this presentation are animated. The mostly do not work on a web view.
    2. 2. What can flow cytometry be used for? <ul><li>Immunology </li></ul><ul><li>Hematology </li></ul><ul><li>Pathology </li></ul><ul><li>Microbiology </li></ul><ul><li>Genetics </li></ul><ul><li>Drug discovery </li></ul><ul><li>Toxicity testing </li></ul><ul><li>Cell culture studies </li></ul><ul><li>Functional studies </li></ul>Clinical and Research <ul><li>Chemical Engineering </li></ul><ul><li>Biotechnology </li></ul><ul><li>Agronomy </li></ul><ul><li>Animal Sciences </li></ul>
    3. 3. CELLULAR ANTIGENS Adhesion Receptors Metabolic cytokines structure enzymes Slide courtesy of Jim Bender T cells B Cells Phenotype: …outward physical manifestation…
    4. 4. Cluster Designations (CD) <ul><li>These are based on the Immunology Workshop an international committee that meets in Boston every few years </li></ul><ul><li>Each antigen that is defined on cells is given a unique number </li></ul><ul><li>Until a final number is agreed, antigens can be designated CDw (w=workshop a tentative designation) </li></ul><ul><li>Here is an example of the possible CDs </li></ul>
    5. 5. Immunofluorescence staining specific binding nonspecific binding Slide from Dr. Carleton Stewart
    6. 6. Direct staining <ul><li>Fluorescent probe attached to antibody </li></ul><ul><li>Specific signal: weak, 3dyes/site </li></ul><ul><li>Nonspecific binding: low </li></ul>Slide from Dr. Carleton Stewart
    7. 7. Avidin-Biotin method I biotinylated primary Ab biotin avidin biotinylated dye
    8. 8. CFU-GM MYELOBLAST MYELOCYTE META- MYELOCYTE BAND PMN CD16 CD11b CDw13 CD33 CD34 HLA-Dr CD38 CD71 MY8 Myelomonocytic Antigen Distribution Purdue Cytometry Labs PROGRANULOCYTE
    9. 9. From Duque et al, Clin.Immunol.News. Negative Positive Decision Tree in Acute Leukemia HLA-DR T CD13,33 CD19 TdT CD10 CD20 Mu B,T AMLL AML T-ALL AML-M3 AUL ? PRE-BI PRE-BII PRE-BIII PRE-BIV PRE-BV CD13,33
    10. 10. What are the principles in flow cytometry? <ul><li>Light scatter ed by a laser or arc lamp </li></ul><ul><li>Specific fluorescence detection </li></ul><ul><li>Hydrodynamically focused stream of particles </li></ul><ul><li>Electrostatic particle separation for sorting </li></ul><ul><li>Multivariate data analysis capability </li></ul>
    11. 11. Concepts <ul><ul><ul><li>Scatter : Size, shape, granularity, polarized scatter (birefringence), structure </li></ul></ul></ul><ul><ul><ul><li>Fluorescence : </li></ul></ul></ul><ul><ul><ul><li>Intrinsic : Endogenous pyridines and flavins </li></ul></ul></ul><ul><ul><ul><li>Extrinsic : All other fluorescence profiles </li></ul></ul></ul><ul><ul><ul><li>Absorption : Loss of light (blocked) </li></ul></ul></ul><ul><ul><ul><li>Time : Useful for kinetics, QC </li></ul></ul></ul><ul><ul><ul><li>Count : Number of cells collected in a histogram </li></ul></ul></ul>
    12. 12. Clinical Analyzers
    13. 13. Cell Sorters (FACS – Fluorescence Activated Cell Sorter)
    14. 14. Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample
    15. 15. Hydrodynamic Systems Sample in Sheath Sheath in Laser beam Piezoelectric crystal oscillator Fluorescence Sensors Scatter Sensor Core Sheath Flow Chamber ++ -- Signal direction
    16. 18. Light Scatter <ul><li>Materials scatter light at wavelengths at which they do not absorb </li></ul><ul><li>If we consider the visible spectrum to be 350-850 nm then small particles (< 1/10  ) scatter rather than absorb light </li></ul><ul><li>For small particles (molecular up to sub micron) the Rayleigh scatter intensity at 0 o and 180 o are about the same </li></ul><ul><li>For larger particles (i.e. size from 1/4 to tens of wavelengths) larger amounts of scatter occur in the forward not the side scatter direction - this is called Mie Scatter (after Gustav Mie) - thus forward scatter is related to size (at 1-15 microns) </li></ul>Shapiro p 79
    17. 19. Optics for forward scatter scatter detector iris blocker Laser beam Stream in air or a round capillary
    18. 20. Frequency distribution histogram Number of events Intensity of parameter (e.g. fluorescence)
    19. 21. histogram Intensity of parameter Number of events
    20. 22. Flow cytometry measurements L M G SCATTER FLUORESCENCE IMAGE
    21. 23. 0 200 400 600 800 1000 Side Scatter Projection Light Scatter Gating Forward Scatter Projection 90 Degree Scatter Neutrophils Lymphocytes Monocytes Forward Scatter Human white blood cells
    22. 24. Different size cells Particle or cell size (log scale) Number of events small large 0.1 1 10 100 1000 0.9 20 90 200 700 While forward light scatter is not always related to cell size, in The majority of cases between 1-12 microns, it is a reasonable estimate
    23. 25. Light Scatter of white blood cells <ul><li>Light scatter can be used to identify populations of cells </li></ul>x In peripheral blood, the three main populations of leukocytes can be distinguished. A “gate” or “bitmap” can be placed around a region so that further analysis can be made on this region. The cells in the region marked “X” can be evaluated as a population.
    24. 26. Fluorescence - e.g. Monoclonal Antibodies
    25. 27. “ B” Cells “ T” Cells
    26. 28. 3 Parameter Data Display Isometric Display
    27. 29. The Cell Cycle G 1 M G 2 S G 0 Quiescent cells
    28. 30. Definitions & Terms <ul><li>Ploidy </li></ul><ul><ul><li>related to the number of chromosomes in a cell </li></ul></ul><ul><li>Haploid : Number of chromosomes in a gamete (germ cell) is called the HAPLOID number for that particular species </li></ul><ul><li>Diploid : The number of cells in a somatic cell for a particular species </li></ul><ul><li>Hyperdiploid : greater than the normal 2n number of chromosomes </li></ul><ul><li>Hypodiploid : Less than the normal 2n number of chromosomes </li></ul><ul><li>DNA Tetraploidy : Containing double the number of chromosomes </li></ul><ul><li>DNA Index : The ratio between the mode of the relative DNA content of the test cells (in G 0 /G 1 phase) to the mode of the relative DNA content in normal G 0 /G 1 diploid cells </li></ul><ul><li>Coefficient of Variation - CV : The ratio between the SD of the mode of the G 0 /G 1 cell populations expressed as a percentage. </li></ul>
    29. 31. Normal Cell Cycle G 0 G 0 - G 1 s DNA Content 2N 4N G 2 M G 0 G 1 s Cell Count G 2 M 0 200 400 600 800 1000 0 75 150 225 300
    30. 32. A typical DNA Histogram G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events 2n 4n
    31. 33. Analyzing the DNA Histogram
    32. 34. Chromosome Analysis Most human chromosomes can be separated by flow cytometry
    33. 35. Chromosome Analysis (Bivariate Flow Karyotyping - porcine) chromosome 1 chromosome 2
    34. 36. log Thiazole Orange .1 1000 100 10 1 Count 0 150 112 75 37 RMI = 0 log Thiazole Orange .1 1000 100 10 1 Count 0 150 112 75 37 RMI = 34 Reticulocyte Analysis
    35. 37. 4 colors - simultaneous collection (can go to 17 colors) Emission wavelength (nm) 530 580 630 680 730 780 FITC PE PE- TR PE-CY5 We separate different subsets by taking bands of light from the light spectrum and analyzing the intensity of light in that band
    36. 38. CD3 CD3 CD3 CD56 CD56 CD8 FOUR COLOR PATTERN CD4 CD8 CD56 - NK CD8 CD4 CD4 Data from Dr. Carleton Stewart CD56 – NK Cells CD3 – T cells CD4 – T cells – Helper CD8 – T cells - Cytotoxic This is a subset of cells It is CD3 + CD56 + This is a subset of cells It is CD3 + CD4 +
    37. 39. Multicolor Analysis Roederer, et al
    38. 40. Cellular Response: <ul><li>Cell death </li></ul><ul><li>Cell ‘suicide’ </li></ul><ul><li>Ignore damage </li></ul><ul><li>Damage repair </li></ul><ul><li>Incorrect repair </li></ul>
    39. 41. Functional Assays <ul><li>intracellular pH </li></ul><ul><li>intracellular calcium </li></ul><ul><li>intracellular glutathione </li></ul><ul><li>oxidative burst </li></ul><ul><li>phagocytosis </li></ul>
    40. 42. TIME (seconds) 0 2400 1800 1200 600 Scale 345 115 38 12 4 Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Unstimulated Neutrophils
    41. 43. FITC-Labeled Bacteria Phagocytosis
    42. 44. Cellular Functions <ul><li>Cell Viability </li></ul><ul><li>Phagocytosis </li></ul><ul><li>Organelle Function </li></ul><ul><ul><li>mitochondria, ER </li></ul></ul><ul><ul><li>endosomes, Golgi </li></ul></ul><ul><li>Oxidative Reactions </li></ul><ul><ul><li>Superoxide </li></ul></ul><ul><ul><li>Hydrogen Peroxide </li></ul></ul><ul><ul><li>Nitric Oxide </li></ul></ul><ul><ul><li>Glutathione levels </li></ul></ul><ul><li>Ionic Flux Determinations </li></ul><ul><ul><li>Calcium </li></ul></ul><ul><ul><li>Intracellular pH </li></ul></ul><ul><li>Membrane Potential </li></ul><ul><li>Membrane Polarization </li></ul><ul><li>Lipid Peroxidation </li></ul>
    43. 45. Organelle Function <ul><li>Mitochondria Rhodamine 123 </li></ul><ul><li>Endosomes Ceramides </li></ul><ul><li>Golgi BODIPY-Ceramide </li></ul><ul><li>Endoplasmic Reticulum DiOC 6 (3) Carbocyanine </li></ul>
    44. 46. Fluorescent Indicators <ul><li>How the assays work: </li></ul><ul><li>Superoxide : Utilizes hydroethidine the sodium borohydride reduced derivative of EB </li></ul><ul><li>Hydrogen Peroxide : DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H 2 O 2 ) oxidize the DCFH to fluorescent DCF </li></ul><ul><li>Glutathione : In human samples measured using 40  M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time. </li></ul><ul><li>Nitric Oxide : DCFH-DA can indicate for nitric oxide in a similar manner to H 2 O 2 so care must be used. DAF is a specific probe available for Nitric Oxide </li></ul>
    45. 47. DCFH-DA DCFH DCF Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H 2 O 2 COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O DCFH-DA DCFH-DA DCFH DCF H O 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence .1 1000 100 10 1 0 20 40 60 counts PMA-stimulated PMN Control 80
    46. 48. Phagosome O 2 O 2 - H 2 O 2 NADPH + H + NADP + HMP NADPH Oxidase GSSG GSH GR GP H 2 O 2 SOD O 2 - H + H 2 O Catalase H 2 O + O 2 PCB SOD PCB (Reduced GSH level) Stimulant PKC PCB (PMA) Human Neutrophil ? ? + O 2 - OH . Lipid Peroxidation Phospolipase A2 activity Leukotrienes H 2 O 2
    47. 49. Cell Sorting <ul><li>Physically separating cells based on some measurable characteristic </li></ul><ul><li>Placing these cells into containers </li></ul>
    48. 50. 488 nm laser + - Fluorescence Activated Cell Sorting Charged Plates Single cells sorted into test tubes FALS Sensor Fluorescence detector Purdue University Cytometry Laboratories
    49. 51. SMALL BEAD LARGE BEAD Frequency Histogram SMALL BEAD LARGE BEAD Sample in Sheath Sheath in Laser beam Stream Charge +2KV -2KV Waste SORT RIGHT SORT LEFT SORT DECISIONS Piezoelectric crystal oscillator Last attached droplet LEFT RIGHT Sensors Sensor
    50. 52. Cell Sorting Video of the droplet formation in a sort stream from a Cytomation instrument. Source: Purdue CDROM vol 4, 1998 Video2.mpg +++ ---
    51. 53. Bindley Bioscience Center Discovery Park
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