J. Philip McCoy, Jr., Ph.D., H.C.L.D. J. Philip McCoy, Jr., PhD

Transcription

1 J. Philip McCoy, Jr., Ph.D., H.C.L.D.

2 1 Introduction 2 How It All Works A very brief description of how a flow cytometer works. Please read it it will help you design your experiments. 3 Understanding the Lingo A few definitions and explanations 4 Non tradition flow cytometry assays 5 Tips in Planning 6 Publishing Your Data Do s and Don ts

3 Chapter 1 The measurement ( metry) of cells (cyto ) as they move in a stream (flow) past a set of stationary detectors. May be performed on a vast array of particles in suspension e.g. cell, beads, bacteria, organelles. Generally requires single particle (cell) suspensions (no clumps or aggregates).

4 In other words, flow cytometry involves analyzing cells (or particles) which have been placed in suspension. These cells, which have generally been tagged with a fluorescent marker of some sort, are then forced into a narrow stream. The stream is intersected by light from one or more lasers, and the fluorescence from the cells is collected by a set of detectors.

5 Flow cytometry may not be the appropriate method for all your needs. The main limitations of flow cytometry are the lack of intrinsic morphologic data, and the inability to analyze cell aggregates or tissue specimens. In situations where this is important, consider using alternative methodologies.

6 Immunohistochemistry if morphology is needed Confocal microscopy for adherent cells and tissues Imaging flow cytometry Combines elements of traditional flow cytometry with imaging PCR Genomic information may prove useful in many circumstances

7 Flow cytometry is a unique methodology that should be considered for use when: Rare event analysis in which large numbers of individual cells need to be analyzed A correlated analysis of as many as 20 parameters of individual cells is desired. High throughput is needed Correlation of cell cycle with immunophenotype

8 Luminex multibead array For soluble analytes such as serum cytokines and chemokines Can measure up to 100 different analytes simultaneously on as little as 50ul of sample.

9 Amnis Imagestream Provides images of all cells in addition to tradition flow cytometry data Technology involved is slightly different than traditional flow cytometry Data acquisition is slower, and no sorting can be performed. Frequency Bright Detail Similarity GFP-Mitotracker Deep Red 3/1367 (0.2%) Not Co-localized Co-localized. 1174/1417 (83%) Co-localized

10 Chapter 2 A Brief Description of How a Flow Cytometer Works

11 To the casual user, the flow cytometer appears to be a complex, somewhat random array of wires, tubes, and gadgets. Remembering our definition of flow cytometry, it is useful to think of a flow cytometer in terms of its organ systems

12 Fluidics (provide the flow) Lasers (provide the light source) Optics (collect the light signals) Electronics (convert the light signal into digital information) Computer (for control of the instrument and interpretation and visualization of data)

13 Necessary for pushing the cells past the detectors. The stream needs to be precisely aligned with laser and optical path. Air bubbles or clumps of cells will alter the stream and effect data. Can be made into droplets for sorting. Sheath stream Excitation laser Sample Fluorescence signal Light Scatter

14 A coaxial stream is used to push the sample past the laser. The outer stream is the sheath fluid (generally isotonic PBS). The inner stream is the sample fluid. A coaxial stream allows the cells to flow with less turbulence, and directs the cells to the center of the stream (hydrodynamic focusing). Sheath fluid Sample fluid Flow Cell Sample (Cells)

15 Emission wavelength of laser must match excitation wavelength of fluorochrome. Argon, Krypton, HeNe are most common lasers. Some lasers can be tuned to different wavelengths.

16 Light fluorescing from the cells must be collected. Fluorescent light emitted from the cells must be distinct and separated from the laser light. In multi color experiments, each color of light must be collected separately. Mirrors, filters, and beam splitters are used to direct and separate the different wavelengths of light. The next figure illustrates how this occurs.

17 Optics of a Clinical Flow Cytometer SSC 488nm/10BP FL1 530nm/30BP FL2 585nm/42BP 90/10 Beam splitter FL4 661nm/16BP DM 560nm SP 670nm LP FL3 Half mirror DM 640LP Fluorescence collection lens 488 nm Blue Laser Red Diode Laser ~635 nm Focusing lens Flow Cell FSC diode 488/10 Modified with permission from BD Biosciences

18 In addition to fluorescence, the cytometer can also collect light scatter measurements. These measurement are how the laser light is scattered by the individual cells. Generally, two types of light scatter are measured: Forward light scatter This is along the axis of laser illumination, and is roughly proportional to cell size, assuming the cell is spherical. Side (orthogonal) light scatter This is general measured at 90 o to the laser path (although it can be at other angles), and provides information concerning the complexity or granularity of cells.

19 Fluorescence signals must be converted to analog pulses (by photomultiplier tubes PMTs), and digitized (by analog to digital converters [ADCs]) for computer analysis.

20 May be used for both control of the cytometer and analysis of data. Requires digitized data for analysis termed listmode files. Analysis of data is possible on computers not connected to cytometer.

21 In order to ensure that all of these systems are operating properly and in concert with each other, rigorous quality control and maintenance procedures are performed on the cytometer. Many of these procedures are performed daily, others at various intervals

22 Much of the quality control involves running beads of various types. These can be used to align the stream/laser/optics with each other or to check that fixed alignment remains accurate. These procedures can take time. Be patient. If the instrument is not set up correctly, your data will be flawed.

23 Practical Flow Cytometry, 4th Edition Howard M. Shapiro ISBN: Pages Dec 2003 $ (or free from Beckman Coulter at This is a VERY technical, but very well-written book. Considered to be the bible of flow cytometry

24 Having this basic understanding of how a flow cytometer works should assist you in preparing and running your samples. Realize that a poorly prepared specimen will give poor results, even with a well maintained cytometer. Similarly, a poorly maintained cytometer will yield poor results for even the best prepared specimen.

25 Chapter 3 A Few Definitions and Explanations

26 As with most areas of technology, flow cytometry has its unique vocabulary. New users of flow cytometry may be confused when asked about gating or compensation or PMTs. Let s demystify some of this.

27 The typical method of viewing flow cytometric data is via univariate or bivariate displays, or histograms. The former displays the fluorescence intensity of the cells along the X axis and the number of cells on the Y axis. The latter, bivariate plots, display the fluorescence intensity of two stains along the X and Y axes. The number of cells is indicated either by dot density or by using contours.

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29 Bivariate Univariate of X axis parameter

30 When analyzing two parameters using a dot plot, lines may be drawn which divide the plot into quadrants. The software will provide statistics on each quadrant, such a the percentage of positive cells, the mean fluorescent intensity of the cells, the coefficient of variation of the staining, and so forth.

31 Wrong Right F L 2 P I FL1 FITC MAb FL1 FITC MAb

32 Gating is a way to tell the cytometer to only examine a portion of the data being collected. This permits heterogeneous cell populations to be put on the cytometer when analysis is only desired on one of the subpopulations. The next slide illustrates this with peripheral blood. Gates are set, or drawn, either during acquisition of data or during analysis (preferred). Multiple gates may be used simultaneously using Boolean logic. Subsequent histograms may be drawn of only the gated cells.

33 Ungated blood Light scatter Corresponding Fluorescence Gated lymphocytes Light scatter Corresponding Fluorescence

34 Multi color flow cytometric experiments are complicated by the fact that fluorochromes often have some overlap in their emission spectra. A small proportion of FITC, which we see as green, will be detected in the orange detector. Similarly, phycoerythrin (PE), which we see as orange, will yield a small signal in the green detector. This is termed bleed over of fluorescence or fluorescence crosstalk.

35 FITC PE

36 How data appear if under-, over-, or properly compensated. Under compensated may falsely appear to be dual positive Proper compensation minimizes on-axis events Over compensated may miss dual positive cells since cells are forced too far to the axis

37 Cluster Designations are system of standardized nomenclature for monoclonal antibodies. These have been established by a series of international workshops. Several hundred CDs have been agreed upon. Several monoclonal Abs may have the same CD, but may recognize different epitopes on the same antigen (e.g. various clones for markers such as CD34 or CD14 differ widely in the epitopes recognized). Thus, not all CD34 Mabs will stain cells identically, nor will all CD14 clones stain cells identically. In general it must not be assumed that all clones with a common CD will stain cells identically.

38 Whenever possible refer to monoclonal antibodies by the CD, not by the individual clone. However, when publishing, including the clone name is recommended.

39 Today, most flow cytometry data are collected as listmode files in.fcs format. This means that information is stored for all parameters measured on each individual cell in a correlated manner. In other words, it is raw data and you can use this to replay experiments, change gates, and sometimes even change compensation.

40 .FCS files are flow cytometry standard files, and represent a somewhat standardized format for the listmode data (although there is some variation between instrument manufacturers in these files)..fcs files can be reanalyzed including regating and, in some instances, resetting compensation values.

41 All flow cytometry requires that cells be forced through a small orifice referred to as a flow cell, or nozzle tip. These vary in size, but for most applications are about

42 A cell 10 microns in diameter flows well through a flow tip with a 70 micron opening. Ten or twelve of these cells in a clump will not.

43 Chapter 4 Beyond Immunophenotyping

44 Flow cytometry has traditionally been used for the study of cell surface markers and for DNA/cell cycle analysis. Flow cytometry can be used for: Intracellular antigens Soluble cytokines or other ligands PCR and FISH Green fluorescent protein transfection Quantitation of antigen expression

45 T cell receptor alpha and beta variable chains. Cytokine receptors. Fetal hemoglobin in fetal red blood cells and f cells. FRET (energy transfer) analysis.

46 Fluorescent particles Phagocytosed by a nonfluorescent cell Will make that cell fluorescent

47 Some drugs (eg adriamycin) will fluoresce at specific wavelengths. Cells which uptake these drugs become fluorescent. Drug uptake or efflux can be monitored (multidrug resistance [MDR] can be assessed this way). No MDR, cells with adriamycin fluoresce MDR pumps out adriamycin Cells become less fluorescent

48 SAC permits analysis of cell free proteins or ligands (such as cytokines) using beads and capture antibodies. As many as 100 spectrally distinct beads analyzed concomitantly, although 5 10 distinct sets of beads is more common. Commercial kits available.

49 Multiplex Bead Array Assays

50 Multiplex Bead Array Assays Beads with different fluorochrome ratios would have distinct spectral addresses in 2 parameter histograms These fluorescent parameters are used for gating beads of interest

51 Flow Cytometry can be used with molecular biology techniques. FISH In situ PCR (eg for detection of HIV) Telomere length measurement GFP or YFP detection

52 Chapter 5 Or How to Keep Your Data Flowing

53 A key to obtaining good data in flow cytometry experiments is the following equation: Garbage in = Garbage Out In other words, a poor specimen or sloppy staining or preparation methods will always yield poor data.

54 Label your tubes!!! Check to make sure that the tube size or microtiter plate that you are using to prepare your samples is compatible with the cytometer CONTROLS,, both positive and negative, will make instrument setup and data interpretation much easier. Compensation controls are needed to accurately set compensation.

55 Assess the viability of your specimen. Nonviable cells can nonspecifically bind antibody and yield confusing results. Trypan blue may be used to manually determine viability, but it is even better to add a real time viability stain to your samples for analysis on the cytometer. If a high proportion of dead cells are found in the specimen, it may be possible to remove them by gradient separation or by simply gating them out if you have performed real time viability staining.

56 An example of a real time viability stain would be propidium iodide (or 7AAD) that stains only dead cells and permits them to be gated out of further analysis. If you use propidium iodide remember that it has a wide emission spectrum and may interfere with other fluorochromes that you plan on using.

57 F L 2 A population with fluorescence like this (dual + at a 45 o angle) is likely due to dead cells P E FL1 FITC MAb

58 The best solution to the problem of cell clumps is to filter samples before they are placed into a cytometer. 50 micron pore nylon mesh tubing is the most often used. Alternatively, large clumps can be removed by allowing them to settle to the bottom of a test tube and removing the cells remaining in suspension. Finally, EDTA may reduce the formation of clumps, although it may be less effective in dissolving them.

59 Flow cytometry is based on the premise of single cell analysis. A cytometer may recognized two cells stuck together as one large cell. This would look like one double positive cell.

60 This population could be a clump of red and green cells F L 2 P I F L 2 P I FL1 FITC MAb FL1 FITC MAb

61 Not all fluorochromes can be run on all flow cytometers. This is partially dependent on which laser(s) light source is on the cytometer. Not all fluorochromes can be run with each other due to spectral overlap. Some fluorochromes are brighter (PE) than others (FITC) (due to better quantum yield). The brighter fluorochromes are better to use if you are expecting dim staining or if you have observed dim staining with other fluorochromes.

62 Fluorochromes which are generally usable: Green FITC or GFP (525nm emission) Orange PE or PI (585nm emission) Red PerCP or PE Cy5 tandem conjugate (680nm emission) Red (with red laser) APC or Cy5 (660nm emission) Many other fluorochromes can be used! Check with the flow lab before staining if you are uncertain.

63 This is one of the most frequently asked questions. There is no one universal answer for this question. It is usually best to estimate the number of cells you need for an assay by asking the following: How rare is the population of interest? Do you have instrument settings or does some of your sample need to be used to set these? What is your cell loss during processing and staining?

64 Chapter 6 Do s and Don ts

65 Checklist: Type of specimen (ATCC #, etc if cell line). Density gradient separation or RBC lysis? Dyes and fluorochromes used. Monoclonal antibodies Conjugations Clone name and CD if applicable Washes and centrifugations. Buffer and fixatives used. Length and temperature of incubations.

66 Checklist: Type and model of cytometer. Laser (and wavelength) used for excitation. Optics for collection of fluorescence. Log or linear amplification? Number of events (cells) collected/analyzed. Data collected gated or ungated?

67 Checklist: Software (and version) used for acquisition. Software (and version) used for analysis. Any smoothing or altering of histograms or plots.

68 In general the compensation values do not have to be published. Notation should be made if figures are presented as compensated data. State if compensation was set before the data were collected or during analysis.

69 Axes should not be labeled Fl1 or Fl2 unless more information is supplied. It is best to label the axes with the fluorochrome and antibody as shown on the right. Always show tick marks to indicate the scale.

70 NEVER rely solely on isotype controls to set markers or assign quadrants in order to determine the percentage of positive cells. Isotype Control Experimental Would you reset the quadrants?

71 Populations of cells should NOT be split unless absolutely necessary. Quadrants can be set based on the clustering of cell populations. This better describes the biology. Right Wrong

72 A good understanding of the basics of flow cytometry will foster good practices in daily work. This must be accompanies by a good understanding of the biology of the cells you are analyzing. For immunophenotyping, understanding antigenantibody interactions is a must! When in doubt, ask for help remember flow cytometry can generate data anyway you run the instrument but GOOD data only comes from GOOD understanding and practices.

73 Practical Flow Cytometry, 4th Edition, Howard M. Shapiro ( Compensation Website: ( Invitrogen flow cytometry tutorial ( player.html) Flow Cytometry - A Basic Introduction, Michael G Ormerod (