1. FLOW CYTOMETRY FACILITY
UNIVERSITY OF CALGARY
FLOW CYTOMETRY FACILITY
‘Efficient and reliable flow cytometry services with the highest standards of quality and productivity’

2. Flow cytometry is a technique for measuring physical and chemical properties of individual cells as they travel in suspension one by one past a sensing point.

3. BASICS OF FLOW CYTOMETRY
The cells in suspension are forced to pass in a fluid stream through a flow cell.
The fluid stream intersects the focus of a laser.
The laser light is scattered and, if the cells are fluorescent, they produce fluorescent signals.
These light signals are then converted to electronic signals (voltages).

4. INSTRUMENTATION
Fluidics
Optics
3. Electronics

5. FLUIDICS
The purpose of the fluidics system is to transport cells in a fluid stream to the laser beam for interogation.

6. OPTICS
The optics system consist of a laser to illuminate the cells in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors (I.e . to resolve different colors).

7. Only one cell should move through the laser beam at a given moment.
The flow of sheath fluid restricts the cells to the center of the sample core for optimal illumination (hydrodynamic focusing).
Only one cell should move through the laser beam at a given moment.
If the light beam and the stream are not perfectly aligned with each other , the cells within the stream will be erratically illuminated and will give off erratic light signals. That’s why it’s important to prevent the poor alignment of the cells with respect to the laser.

8. “THE LASER”
Lasers emit coherent light, in a fine, straight beam at a specified wavelength.
The use of a laser allows the beam of light to be focused on single cells so that basic measurements based on beam disturbance can be taken (FSC, SSC) .
LASER: Acronym for
Light Amplification by
Stimulated Emission of
Radiation

9. -The voltages are processed by the computer.
ELECTRONICS
Processing of signals from detectors
-Cells passing through the laser beam generate light signals.These light signals are then converted to electronic signals (voltages).
-The electrical voltage generated will be proportional to the number of photons (amount of light) emitted by the cell/particle.
-The voltages are processed by the computer.
Detectors have voltages applied to them so that the cascade of electrons resulting from the original light impulse can be converted into a sufficiently large current to be measured.

10. The sample is injected into a stream of sheath fluid within the flow chamber; the sample core remains separate but coaxial within the sheath fluid.
The sample is injected into the center of a sheath flow. The combined flow is reduced in diameter, forcing the cell into the center of the stream. This schematic of the flow chamber in relation to the laser beam in the sensing area.

11. Detected along the axis of incident light
Light is bent (diffracted) depending on the size and refractive index of the cell
Detected along the axis of incident light
(0-100)
The degree to which the light is bent is measured as FSC.

12. Light is reflected/bounced to the side
Proportional to cell granularity
Detected at 90o to incident light axis

13. When cells pass through the laser intercept, they scatter laser light and the system measures the degree and direction of scattered light-indicators of the cell’s size, shape and structure.

15. What do the scatter signals tell us?
Together the forward and side scatter signals can provide useful ways to characterize different cell types.
Example: Leucocytes (white blood cells).

17. FLUOROSCENCE
Fluorescent compounds absorb light energy over a range of wavelengths, which causes an electron in the compound to be raised to a higher energy level.
The excited electron emits this excess energy as a photon of light.
While each fluorochrome will have an optimal, or peak emission wavelength, the spectra will actually be distributed over a number of wavelengths.

18. Any fluorescent molecules present on the cell fluoresce.

19. Injector
Tip
Sheath
fluid
Fluorescence
signals
Focused laser
beam
The cells are forced to pass in a fluid stream through a flow cell where the fluid stream intersects the focus of the laser.

20. Detector
Fluorochrome
Emission
FL1
FITC, GFP, Alexa 488, CFSE, Fluo 3
525 nm
FL2
PE, PI
575 nm
FL3
PER-CP, Cychrome, 7-AAD nm
More than one fluorochrome can be used simultaneously if each is excited at 488 nm and if the peak emission wavelengths are not extremely close to each other.

21. Histogram: Relative fluorescence vs. # of events
DATA DISPLAY
Negative Control
Sample
Histogram: Relative fluorescence vs. # of events

22. Quadrants can be applied to any 2 parameter display.
DATA DISPLAY
Quadrants can be applied to any 2 parameter display.

23. IMMUNOPHENOTYPING
Antibody-fluorescent dye conjugates bind to antigens and the quantity of the fluorescent light emitted is correlated with the cellular marker in question.

24. DATA DISPLAY Quadrants can be applied to any 2 parameter display.
You can correlate one parameter to another.
Divide the area into 4 discreet populations

25. Activation: Surface Receptor Expression
Up Regulation of IL-2 Receptor on Mouse B Cells

26. CELL ADHESION MOLECULES
A. Unstimulated
--- Isotype
__ Anti-P-Selectin
B. Thrombin-activated
Human peripheral blood platelets were activated with thrombin, stained with either an isotype control or P-selectin-PE antibody and analyzed with flow cytometry. P selectin binds to Pselectin ligand. Psgl-1expressed by neutrophils, monos lymphos.
Expression of P-selectin is up-regulated on activated peripheral blood platelets.

27. Activation: Intracellular Cytokines

28. INTRACELLULAR CYTOKINE MEASUREMENT

29. INTRACELLULAR CYTOKINE MEASUREMENT
Helper T cells secrete cytokines upon activation. There are at least 3 types of T cells (CD4) defined by their pattern of cytokine production. Th1 cells primarily produce IL2 and IFN Gamma Th2 cells make IL4 and IL5 while Th0 cells have a less differentiated cytokine profile. These subsets are not easily distinguishable by their cell surface antigens.
Recently, The ligand for P selectin was reported to discriminate bw Th1 and
Th2 cells.
As shown in figure, spleen cells grown in culture conditions that promote either Th1 or Th2 type cytokine production have a characteristic phenotype with respect to their cytokine production.
Multi-parameter flow cytometric analysis of cultured Th1 and Th2 cells

30. APOPTOSIS

31. METHODS FOR DETECTING APOPTOSIS:
Annexin V assay

32. Apoptosis: Annexin V Assay
Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody

33. PI vs. ANNEXIN
One of the more common uses of DNA-based dyes is to identify apoptotic cells.
Necrotic cells are widely permeable to a number of cell labels (usually PI) whereas apoptotic cells are impermeable.
Staining for apoptotic markers (i.e., Annexin V) will identify apoptotic cells, whereas necrotic cells will also stain with PI.

34. PI vs. ANNEXIN

35. Apoptosis: Quantitative Analysis of Caspase-3 Activation
Jurkat cells Treated with Campothecin
Caspase 3 PE

36. Apoptosis: TUNEL Assay
Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody

37. Apoptosis: bcl-2 Regulation
Mitochondrial Protein bcl-2
Blocks Apoptosis
Bcl-2 Is Down Regulated
During Apoptosis

38. METHODS FOR DETECTING APOPTOSIS:
Gene regulation
Analysis of p53 expression in SV40 transformed rat ovarian cells

39. METHODS FOR DETECTING APOPTOSIS: Histone Phosphorylation
Jurkat cells were treated with staurosporine for the indicated time.

40. Proliferation: Nucleotide Analogs
Bromodeoxyuridine Is
Incorporated Into Cellular DNA
By Pulsing Proliferating Cells
The Nucleotide May Be
Conjugated With Fluorochrome
Or Detected By Antibodies

41. PROLIFERATION: CFSE Carboxyfluorescein succinimidyl ester
The amount of CFSE in the cell in the
membrane of proliferating cells
halves with each successive
division and therefore, the fluorescence can be used to monitor the number of cell divisions.
Cells can be removed from an animal, stained with a fluorochrome which is not harmful to cell function and then reinjected into a donor animal so that the pathways of cell movement can be followed. CFSE which is cleaved by nonspecific esterases after entering the cell and thereafter remains in the cytoplasm of the cell for days to months. CFSE is a lipophilic dye that is taken up by the cell membrane. If cells are labeled with CFSE and then induced to proliferate the level of fluorescence in each daughter cell will be half as much as the parent cell after each cell division. Therefore the fluorescence can be measured and used to monitor the number of cell divisions.
By using CFSE as a dye for tracking cell proliferation, one can select additional parameters (CD markers or intracellular cytokines) and perform further flow cytometric analysis to characterize the nature of cells.
It binds to free amine groups of cytoplasmic macromolecules. Then esterases remove the carboxyl groups converting non fluorescent CFSE to fluorescent. Upon cell division, the CFSE will be uniformly distributed between the daughter cells. CFSE diffuses freely into the cellsand IC esterases cleave the acetate groups converting it to a fluorescent membrane impermeant dye. It retains inside the cell and it does not interfere with the cellular functions.

42. Ploidy: Nucleic Acid Dyes
DAPI is one of many
non-vital dyes that binds DNA on an equimolar basis
This allows the precise
quantitation of a cell
populations proliferative
index

43. DNA & CELL CYCLE ANALYSIS
PI: Key feature of DNA probes is that they are STOICHIOMETRIC.
Total nuclear content and the fraction of cells in each phase of the cell cycle can be measured.
The measurement of the DNA content of cells was one of the first major applications of flow cytometry and is still one of the biggest applications today.
The DNA content of the cell can provide a great deal of information about the cell cycle. In DNA content analysis we are evaluating the relative DNA content of the nucleus of the cell. The fluorochrome most commonly used for this analysis is PI.PI binds to DNA in cells at all stages of the cell division cycle and the intensity with which a cell’s nucleus emits light is directly proprtional to its DNA content.
N=23 2n=46
PI stains all double stranded regions of both DNA and RNA by intercalating bw the stacked bases of the double helix. PI is used with Rnase and it’s not able to penetrate to an intact membrane therefore the membrane should be permeabilized with alcohol.

44. s s Normal Cell Cycle M G2 G2 M G0 G1 G0 G1 DNA Analysis 2N 4N
Count s G2 M 2N 4N
200
400
600
800
1000
DNA content

45. INTRACELLULAR CYTOKINE MEASUREMENT
The production of cytokines by specific cell types can be determined as opposed to measuring the amount of secreted cytokine present in the serum or supernatant.
Intracellular cytokine staining is a method which allows one to take a snapshot in time of the protein content of a single cell. The aim is to make use of the rapid flow cytometric analysis of cells to provide both surface phenotype and intracellular cytokine content of single cells within a population of cells responding to antigenic or mitogenic stimulation. The technique initially requires fixation of cells in paraformaldehyde, which cross-links proteins and prevents their leakage. Secondly, permeabilization of cellular membranes with a detergent generates gaps in the membrane, allowing specific cytokine antibodies access to the cells interior.
Adv: Understanding which cytokines play a key role in immune regulation as well as identifying their cellular origin is currently under intense investigation. Studies to date have made use of cloned cells as developmental tools in elucidating the cytokine secretion pattern of selected cell populations. This information has largely been gained through such methods as Northern Blot, in situ hybridization, RT-PCR and ELISAs. These methods suffer from the disadvantage that they are time consuming, labor intensive and in some cases they can only give information about the properties of the entire population of cells being analyzed. In contrast, intracellular cytokine staining of cells and subsequent flow cytometric analysis can yield rapid and specific informationon the cytokine production properties of individual cells within a mixed population of cells.

46. APOPTOSIS: SUBGENOMIC DNA PEAKS
Frequently, the end point of the apoptotic process is double strand scission of the DNA at the linker regions between the nucleosomes. During fixation of cells some of the lower MWt fragmentsleach out of the cell. Lowering the DNA content. These cells can be observed on a DNA histogram as a hypodiploid or SubG1 peak.
The sub G1 method relies on the fact that after DNA fragmentation, there are small fragments of DNA that are able to be eluted following washing in PBS. This means that after staining with a quantitative DNA binding dye, cells that have lost DNA will take up less stain and will appear to the left of the G1 peak.

47. TRANSFECTION EFFICIENCY
Using Green Fluorescent Protein (GFP) as a co-transformation marker is one of the most common applications of GFP-expressing vectors
Transfection is the introduction of foreign DNA into a eukaryotic cell and it is an important tool for studying the regulation of gene expression as well as protein function. In stable transfection, the foreign DNA becomes integrated into the genomic DNA of the cell so that it is passed on in the cell lineage and continues to express the gene of interest. In order to determine the percentage of the cells that are expressing the foreign DNA sequence, a reporter gene can be used. A convenient reporter for monitoring transfection efficiency with the flow cytometer is the GFP. When excited by blue light cells expressing GFP emits bright green fluorescent light which then can be detected by FACS analysis.
Histogram shows 59% of the cells expressing the peptide and indicates various levels of expression due to non-synchronous expression. Ed outline overlay indicates the background control.

48. CELL NUMBER FLUORESCENCE TRANSGENIC CELL LINE CONTROL
FLUORESCENCE

49. ESTIMATING CELL VIABILITY
PROPIDIUM IODIDE (PI)
-Excluded by viable cells and when taken up by dying cells, binds to nucleic acids and fluoresces orange.
Traditionally, the number of live cells in a suspension is estimated by counting microscopically the cells which exclude an acidic dye such as TB.
This method can be adapted for the flow cyt. By using PI which is excluded by viable cells and when taken up by dead or dying cells, it binds to nucleic acids and fluoresces orange.
Adv: Large number of cells can be counted quickly and the determination of +ve vs -ve is objective.

50. Viability: Fluorescein Diacetate & PI
FDA converted to fluorescent compound in live cells

51. Oxidative Burst
Conversion of nonfluorescent dichlorofluorescein diacetate to
the fluorescent compound 2',7'-dichlorofluorescein can be used
to monitor the oxidative burst in polymorphonuclear leukocytes.

52. CALCIUM FLUX MEASUREMENTS
Many stimuli can cause mobilization of calcium either as an influx from the extracellular medium or the release of intracellular stores.
A number of dyes that fluoresce when bound to calcium are used to “pre-load” cells prior to calcium mobilization and the cells will “glow” as calcium streams into the cells.
Calcium is a very important cellular ion. It plays a vital role in the transduction of signals from the cell membrane to the cell cytoplasm and a change in intracellular calcium levels is a good indication the cell is responding to a stimulus. Many stlimuli can cause mobilisation of calcium either as an influx from the extracellular medium or the release of intracellular stores. By using fluorescent dyes this mobilisation can be observed.

53. Fluo-3 is a Ca+2 sensitive fluorescent probe
Fluo-3 is a Ca+2 sensitive fluorescent probe. Its emission intensity changes with the Ca+2 concentration.
Fluo-3 is nonfluorescent without calcium. It is easily incorporated into cells as an ester at 37o. Once inside the cell, the esters are hydrolyzed by esterases present in the cytosol and remain trapped inside. For the above experiment, fluo loaded unstimulated cells were recorded, followed by the addition of a stimulus.

54. CALCIUM INFLUX
Ca++ Bound Indo-1 at 390 nm to Free Indo-1 at 495 nm

55. PHOSPHOPROTEIN PROFILING WITH FLOW CYTOMETRY
Flow cytometry requires only a small sample size and is ideal for performing quantitative, multiparameter anlayses of single cells or of distinct cell populations. Flow Cytometry is not only rapid and sensitive but it enables the differential evaluation of intracellular signaling events in complex cell populations such as PBMCs.

56. Signal Transduction: Phospospecific mAbs

57. Signal Transduction: Phosphospecific mAbs

58. PHAGOCYTOSIS PHAGOCYTOSIS CAN BE STUDIED USING FLUORESCENT
LATEX OR POLYSTYRENE BEADS. BEAD CAN BE ADDED TO
TISSUE CULTURE OR IN VIVO. AS CELLS TAKE UP THE
FLUORESCENT BEADS, THEY BECOME INCREASINGLY
MORE FLUORESCENT IN AN INCREMENTAL MANNER.
THEY ALSO INCREASE THEIR SSC SINCE THE BEADS INCREASE
THE GRANULARITY OF THE PHAGOCYTES.
This test allows the quantitative determination of leukocyte phagocytosis. It contains FITC labeled opsonized E.coli. The control remains on the ice and the other 37.

59. BD QUANTIBRITE SYSTEM
Calibration beads with known numbers of PE molecules per bead provide an easy and sensitive means of quantifying PE stained cells with a flow cytometer. Quantibrite PE is a lyophilized pellet of beads conjugated with 4 levels of PE, these are used to calibrate the FL2 axis of a flow cytometer in terms of the numbers of PE molecules. When used in conjunction with PE conjugates with a known ratio of PE to antibody, PE molecules can be converted to antibodies bound per cell.
A standard quantitative methodology for defining levels of marker expression is especially important
when characterizing populations that express heterogenous levels of antigen. In addition, activated cells expressing various levels of cytokine detected by intracellular staining techniques represent kinetically discrete populations defined by the amount of accumulated cytokine. Results are often expressed in relative terms such as bright and dim fluorescent staining. These are qualitative terms and the interpretations can vary from lab to lab. On the other hand fluorescence quantitation or the expression of staining intensity levels in terms of absolute numbers of fluorophore molecules per cell can provide meaningful classification of various staining patterns. Traditional analysis of flow cytometry data yields info about the frequency of cells expressing signature antigens. Positive populations are identified as the number of cells that express levels of antigen above isotype control fluorescence. Data are usually reported as a percentage of cells positive or negative for gated populations or as mean fluorescence intensity on a relative scale.
e.g CD69 expression in HIV patients. They have diminished response to mitogens ABC for normal and 5100 for HIV+
A method used to calculate the amount of antibody bound to a cell that correlates to the number of antigens expressed on the cell surface.

60. BD CYTOMETRIC BEAD ARRAY SYSTEM
WASH
The BD CBA employs a series of particles with discreet fluorescence intensities to simultaneously detect multiple soluble analytes from serum plasma or tissue culture supernatant. Efficient capturing of analytes via suspended particles coated with distinct capture antibodies enable the BD CBA to use fewer sample dilutions to determine analyte concentration in substantially less time.
The specific capture beads are mixed with PE cnjugated detection antibodies and then incubated with recombinant protein standards or test samples to form sandwich complexes. Following acquisition, the sample results are generated using the BD CBA analysis software.
Sample values are extrapolated by comparison against a known standard. Conc vs MFI
1. Add unknowns or standards to capture bead array
2. Add detection reagents and incubate
3.Acquire samples

61. Cell Sorting
The population of interest can be separated during flow and deposited into a tube for later analysis (>95% purity).
Any combination of analytical parameters can be used to set the criteria for sorting as opposed to single parameter methods (I.e. separating the negatives from the positives).
The ability of flow cytometers to sort cells specifically identified during analysis adds a further dimension to the capability of these instruments.
While other sorting systems have been developed such as magnetic bead separation, these are generally single parameter methods. Like separating positives from the negatives.
Flow has the major advantage that any combination of any analytical parameters can be used to set the criteria for sorting.

62. Fluorescence detector
Fluorescence Activated
Cell Sorting
488 nm laser
FALS Sensor
Fluorescence detector - +
Charged Plates
Cell Sorting
Some flow cytometers are also equipped to separate and collect user-specified single cells from a sample.
With these instruments ,
the flow cytometer nozzle is vibrated at a high frequency by a piezoelectric transducer t
hat causes the microscopic fluid stream exiting the flow chamber to break into
discrete droplets. As a cell of interest reaches the droplet break-off point, it receives a positive or negative charge.
As the droplets pass individually through two vertical deflection plates,
the electric field created by those plates directs them toward the appropriate, user-specified collection receptacles.
Uncharged droplets flow into a waste receptacle.
Particles exit the flow chamber in a jet which breaks up into regularly spaced droplets. Specific droplets containing the cells of choice are charged and passed through a high V electrostatic field, thus being deflected for collection. Pressurized sheath fluid exits the flow chamber thru an accurately drilled hole. Apply some vibration of known frequency-breaks up into droplets-stable drops at predictable distances from each other. Apply voltage to the droplets-there is a net charge on the droplet. Droplet enters the electromagnetic field formed bw high voltage plates and charged droplets attarcted to the opposite poles, and therefore separated. It’s possible to apply a +ve or a _ve charge to droplets and then cells can be sorted to the left or right of the undeflected stream. Sorting decision is made right after their analysis in the quartz cuvette.
Single cells sorted
into test tubes

63. CELL NUMBER FLUORESCENCE TRANSGENIC CELL LINE POST-SORT
FLUORESCENCE

64. ADVANTAGES
-Evaluating large number of cells quantitatively and reproducibly increases the statistical confidence and precision of data. -The ability to measure several parameters on thousands of single cells within minute (10,000 cells/second). -Measurements are made separately on each cell. -Simultaneous, multiparameter analyses in complex cell populations.

65. The power of flow cytometry lies in the ability to measure several parameters on thousands of single cells within minutes (1000 cells/second).

66. BD FACScan is a 3-colour, fixed alignment bench top analyzer, equipped with a 488 nm air-cooled argon laser. It is capable of simultaneously measuring and analyzing FSC, SSC, and 3 spectral regions of fluorescence.

67. BD LSR: Expands the range of multicolor analysis applications you can run on a benchtop cytometer, with up to six fluorescence and two scatter parameters.
The LSR also provides high resolution DNA ploidy and pulse processing technology that allows the measurement of area, width and ratio of detector pulses.

68. BD FACS Vantage SE: The FACS Vantage SE is an analytical flow cytometer with sorting capabilities. This instrument has 6-color capability and can sort up to 20,000 cells/sec. The FACS Vantage is equipped with high speed and turbo sorting, as well as pulse processing and automated single-cell deposition unit.

69. U OF C FLOW CYTOMETRY USER FACILITY
We offer individual training on
basic flow cytometry on an
BD FACScan (488 laser)
Future courses will include:
Analysis of flow cytometric data
FLOWJO
Measurement of Apoptosis
DNA and Cell Cycle Analysis
For more information please contact Laurie Kennedy ( )

70. PROVIDING FLOW CYTOMETRY & CELL SORTING SERVICES FEATURING THE MOST ADVANCED FLOW TECHNOLOGY
Multi-color immunofluorescence
DNA/Cell cycle analysis
Intracellular cytokine measurements
Fluorescence quantitation
Multi-color, rare event and single cell sorting
Comprehensive computer analysis
Flow Cytometry Core Facility, The University of Calgary Rm Hospital Drive NW, Calgary, AB T2N 4N1 tel

71. PERSONNEL Director: John D. Reynolds (reynolds@ucalgary.ca)
Operator: Laurie Robertson, HSC 2580
Tel:
Operator and Instructor: Laurie Kennedy, HSC 2802
Tel:

72. Please visit our website: