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The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford

¹ Stanford University, Department of Genetics, Stanford, CA 94305.

² The Baxter Laboratory, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305.

³ Vaccine Research Center, NIH, Bethesda, MD 20892.

^aAuthor for correspondence. E-mail lenherz{at}darwin.stanford.edu.

Abstract

The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8–12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.

Preface

I want to thank the AACC for honoring me with the Ullman award and allowing me to present a lecture on the history and future of flow cytometry, which began when my colleagues Richard Sweet, Bill Bonner, Russ Hulett, and I invented the Fluorescence Activated Cell Sorter (FACS). ¹ This honor acknowledges the now enormous field of flow cytometry and its many applications in immunology, cell biology, and molecular biology, and its clinical applications in AIDS, leukemia/lymphoma, and cancer detection and analysis, as well as clinical chemistry. Fittingly, the original support for work that led to the FACS came from the NIH under a grant program called "Automating the Clinical Laboratory" beginning in the late 1960s.

Introduction

I have attempted here to reproduce the Ullman Award lecture entitled "Past, Present and Future of Fluorescence Activated Cell Sorting (FACS)" that the AACC so graciously invited me to present.

In Fig. 1 , I show a diagram of the FACS from our 1974 article in Scientific American (1), which shows one laser and two light detectors, one for forward scatter (a measure of cell size) and one for fluorescence. The analysis and sorting essentials of the FACS are unchanged to this day—quite unusual for a high-tech instrument, particularly one that is used in thousands of laboratories throughout the world. A recent estimate of the number of FACS instruments (generically defined) is 30 000 analyzers and sorters.

View larger version (127K): [in this window] [in a new window]   Figure 1. Diagram of the FACS from our 1974 article in Scientific American, showing one laser and two light detectors, one for forward scatter (a measure of cell size) and one for fluorescence.

The Past

The ability to analyze and sort live cells was an important feature of our first machines. This made many of the applications developed in our laboratory and elsewhere possible. Particularly, it enabled determination of the functions of subsets of the cells that coexist in blood and various organs. I will show some illustrations of these studies later.

Shown in Fig. 2 is a diagram of a modern three-laser sorter with multiple detectors. I will show later that we now simultaneously detect 12 different fluorescent colors and have considerably increased the speed of analysis and sorting. Several of our collaborators have given different names to this modern sorter capability. We currently refer to it as "Hi-D FACS".

View larger version (111K): [in this window] [in a new window]   Figure 2. Diagram of a modern three-laser sorter with three lasers and multiple detectors.

Stroboscopic images, photographed many years ago, of the stream and droplets coming from the FACS nozzle are shown in Fig. 3 . Thousands of droplets are dramatically superimposed in each image, kept in place by the fantastic constancy of the stream, droplet generation, and droplet separation that characterizes the FACS operation. Individual cells, identified as they pass through the illumination/detection zone before the stream breaks up into droplets, are passively carried in only a minority of the droplets. Droplets containing cells that meet the sort criteria are charged at the moment of formation, and the charged droplets are sorted as they pass between constantly charged deflecting plates. This type of droplet separation was originally applied by Richard Sweet in his invention of the "ink-jet printer".

View larger version (73K): [in this window] [in a new window]   Figure 3. Stroboscopic images, photographed many years ago, of the stream and droplets coming from the FACS nozzle. In these time-lapse photographs, many droplets are superimposed, showing the constancy of drop formation.

Shown in Fig. 4 is a beautiful old photo of laser beams under a nozzle and the deflection plates with an array of culture wells below.

View larger version (53K): [in this window] [in a new window]   Figure 4. Beautiful old photograph of laser beams under a nozzle and the deflection plates with an array of culture wells below.

More Fluorescence Colors

One of the early recognized needs was for more fluorescent colors so that we could use more reagents to simultaneously detect markers on cells. We started with two colors, fluorescein and rhodamine; later, Texas red was substituted for rhodamine. We found the first of the additional fluorescence colors in seaweeds off the local California coast. When you shine ultraviolet light on these seaweeds, you see a beautiful fluorescent picture (Fig. 5 ). We [Vernon Oi, Randy Hardy, and others (2)] separated the pigments by chromatography. The pigments are called phycobiliproteins. The patent for the phycobiliprotein uses in FACS and other applications was issued to Glaser, Oi, and Stryer (3). Our laboratory pioneered the initial uses of these versatile and now widely used proteins in FACS studies.

View larger version (146K): [in this window] [in a new window]   Figure 5. Beautiful fluorescent picture produced when ultraviolet light is shown on seaweeds. The phycobiliproteins are antenna molecules with tens of fluorochromes that transfer energy from wavelengths available underwater to chlorophyll so the plants can grow.

Green fluorescent protein (GFP) is another example of a fluorescent protein that is widely used in FACS studies. GFP is made by the jelly fish Aequoria victoria (4) (Fig. 6 ) and is spontaneously fluorescent. The gene coding for GFP has been cloned and is now widely used as a reporter gene because the extent of its expression is readily measured by FACS.

View larger version (83K): [in this window] [in a new window]   Figure 6. GFP is made by the jellyfish A. victoria and is spontaneously fluorescent.

We [Michael Anderson et al. (5)] mutated GFP to make two forms, which are excited at different wavelengths but emit at a common wavelength. In a multiple-laser FACS, in which the laser beams that excite each of the GFP forms intersect the cell stream one after the other, the signals emitted by each form are easily distinguished because they are directed to different detectors and arrive at different times. This effectively provides two reporter gene signals detectable from the same cell. Spectra of the violet excited (Vex) and the blue excited (Bex) mutants, as well as wild-type GFP, are shown in Fig. 7 .

View larger version (48K): [in this window] [in a new window]   Figure 7. Illustration of the violet excited (Vex) and the blue excited (Bex) mutants, as well as wild-type GFP.

FACS and Monoclonal Antibodies: Complementary Tools

The discovery that really made the FACS and flow cytometry a laboratory and clinical standard was the production of hybridomas by Kohler and Milstein (6)(7). As is commonly known, hybridomas produce unlimited amounts of distinctive monoclonal antibodies, each of which is highly specific for its target antigens and can readily be coupled to fluorescein, phycobiliproteins, and other fluorochromes. The use of monoclonal antibodies as FACS reagents, which our laboratory pioneered, has enabled definition of hundreds of target antigens present on or in cells.

The FACS not only uses monoclonal antibodies produced by hybridomas, it is commonly used during hybridoma development to sort and clone individual cells from a hybridoma fusion. Single-cell sorting with the FACS is the most efficient way of cloning cells, especially when they are present at a very low frequency. In fact, many a hybridoma clone has been rescued by FACS from cultures where nonproducers were about to overgrow and a monoclonal antibody was about to be lost forever (8).

The FACS Gal Reporter Gene System

An example of FACS use in functional assays is shown in Table 1 . Some years ago, we (Garry Nolan and I) developed FACS Gal (9), an intracellular ß-galactosidase reporter gene assay system for studies of somatic cell genetics and gene regulation.

Hi-D FACS

With FACS Gal, phycobiliproteins, GFP, and monoclonal antibodies that could detect a wide variety of surface markers, we began to need and justify increasing the number of colors that could be simultaneously measured by FACS. Over the next few years, we published several reports on progressively increasing the number of colors and applying the progressively powerful FACS in new applications (1)(5)(10)(11)(12)(13)(14)(15).

Currently, we have a 12-color FACS that detects the colors shown in Fig. 8 . Three lasers are needed to excite this many colors, indicated by the colored bars on the right for the krypton, argon, and dye laser, respectively. The different dyes are indicated on the left, whereas the excitation and emission spectra, as well as the laser line wavelengths, are given in the body of Fig. 8 . The emission filters are indicated by the gray bars, which indicate both the central wavelength and the range of wavelengths passed by the collection filters. I summarize some of the advantages of using this instrument in Table 2 .

View larger version (42K): [in this window] [in a new window]   Figure 8. Twelve-color FACS that detects the colors shown. Three lasers are needed to excite this many colors, indicated by the colored bars on the right for the krypton, argon, and dye lasers, respectively. The different dyes are indicated on the left, whereas the excitation and emission spectra, as well as the laser line wavelengths, are given in the body of the figure. The emission filters are indicated by the gray bars, which indicate both the central wavelength and the range of wavelengths passed by the collection filters. PE, phycoerythrin; TRPE, Texas red phycoerythrin; APC, allophycocyanin.

Our present studies focusing on functional distinctions between subsets of human peripheral blood cells include studies of cytokine production by individual T-cell subsets, expression of activation markers, and apoptosis induction in such subsets (16). Some of the methods we have developed for this work are outlined in Tables 3 and 4 .

The importance of ethidium monoazide (EMA) staining for getting accurate cytokine measurements is illustrated in an example of CD4 cells making two cytokines (Fig. 9 ). Another example of the importance of EMA is staining for intracellular kinases, the protocol for which is given in Table 5 . Fig. 10 shows color density and dot plots of intracellular staining, illustrating the importance of using EMA, which covalently and stably labels dead cells, to distinguish the dead cells before fixation and permeabilization when staining for intracellular kinase activity. Fully 23% of the stimulated cells harvested for staining were dead, as judged by EMA staining. The lower left plot shows the percentages of cells in which three kinases are detected after gating out the dead cells.

View larger version (53K): [in this window] [in a new window]   Figure 9. The importance of EMA staining for getting accurate cytokine measurements in an example of CD4 cells making two cytokines. PMA, phorbol myristate acetate; FS, forward scatter; IFN, interferon-; IL-4, interleukin-4.

View larger version (40K): [in this window] [in a new window]   Figure 10. Color density and plots of intracellular staining. This figure illustrates the importance of using EMA, which covalently and stably labels dead cells, to distinguish them before fixation and permeabilization in staining procedures for intracellular kinase activities. FSC, forward scatter.

Redox Studies by FACS

We now turn to some Hi-D studies that focus on redox, a major current interest of our laboratory. We have used Hi-D FACS to examine intracellular redox status in subsets of peripheral blood mononuclear cells (PBMCs) from individuals with HIV infection, inflammation, sepsis, and other disease (16)(17)(18)(19)(20). In the first example, shown in Fig. 11 , we measured intracellular glutathione (GSH) concentrations in CD4 T cells and other PBMC subsets in HIV-infected individuals. Results showed that GSH concentrations, detected by FACS as glutathione S-bimane (GSB), are correlated in PBMC subsets (20).

View larger version (67K): [in this window] [in a new window]   Figure 11. Measured intracellular GSH concentrations in CD4 T cells and other PBMC subsets in HIV-infected individuals. Results show that GSH concentrations, detected by FACS as GSB, are correlated in PBMC subsets.

Two recent examples, not yet published, are shown in Figs. 12 and 13 . In one, we used another fluorescent Molecular Probes reagent, Alexa594 maleimide (ALM), which covalently binds to -SH groups and thus provides a measure of cell surface thiols on viable cells. In the second, we used a monoclonal antibody to a well-known redox molecule, thioredoxin, to measure intracellular thioredoxin concentrations. Fig. 13 shows the positive correlation between surface thiols and intracellular thioredoxin: both measures show high concentrations in HIV-infected individuals and lower concentrations in healthy controls. When we complete this study, we hope to understand more about redox regulation and the relationship of intracellular redox and surface thiols.

View larger version (17K): [in this window] [in a new window]   Figure 12. Use of a fluorescent Molecular Probes reagent, Alexa maleimide (ALM), which covalently binds to -SH groups and therefore provides a measure of cell surface thiols on viable cells.

View larger version (21K): [in this window] [in a new window]   Figure 13. Positive correlation between surface thiols and intracellular thioredoxin (iTrx). Both measures show high concentrations in HIV-infected individuals and lower concentrations in healthy controls. mfi, median fluorescence intensity.

Where to Now?

This leads us to what I think we are all interested in: where is FACS going? This broadly useful technology, which started in the late 1960s and became more and more widely used through the 1990s, has been evolving slowly throughout this period. We plan to see that it continues to evolve in this new century and perhaps to help speed up evolution to the benefit of all FACS users. Tables 6 and 7 outline some of the goals for the improvement of FACS and the application of new FACS methods for cell analysis and disease diagnosis and monitoring.

In this short review of work primarily from the Herzenberg Laboratory, students and postdoctorate fellows from the laboratory, and collaborators, we have not mentioned many of the people who have made important contributions to flow cytometry. Early contributors to the field include Mack Fulwyer and Marvin van Dilla from Los Alamos National Laboratory (21). A very important documenter and innovator is Howard Shapiro with his several editions of Practical Flow Cytometry (22).

View larger version (31K): [in this window] [in a new window]   Figure 14. Acknowledgments.

Acknowledgments

This work has clearly been the labor and ideas of many students, postdoctorate fellows, engineers, and others who have passed through the Herzenberg Laboratory in the last three to four decades. Some are included by name, but many are not listed. In Fig. 14 those whose work is specifically mentioned are shown more clearly.

Footnotes

¹ Nonstandard abbreviations: FACS, fluorescence activated cell sorter; GFP, green fluorescent protein; EMA, ethidium monoazide; PBMC, peripheral blood mononuclear cell; GSH, glutathione; and GSB, glutathione S-bimane.

References

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