Leonard Herzenberg
The Fluorescence-Activated Cell-Sorter (FACS) sorts cell by putting a tiny electrical charge on a droplet carrying a desired cell, and then passes that through an electric field. Charged droplets move left or right, and fall into collection tubes down below. Uncharged droplets, which are empty or contain unwanted material, fall in to the waste collection vessel. Expansion on FACS capabilities have included the cloning of hybridoma cells, adding more fluorescence colors and developing the dual-laser machine, using ethidium monoazide (EMA) to identify dead cells, and improving the hardware and FACS software.
Genetics, FACS, Immunology, and Redox: A Tale of Two Lives Intertwined
Leonard Herzenberg, and Leonore Herzenberg
In this paper, Dr. Len and Lee Herzenberg detail their journey through academia, from their days at Caltech, to the Pasteur Institute in Paris, to the NIH, and finally to Stanford. They discuss some of the most influential researchers with whom they have cooperated. Caltech's environment fostered scientific exploration along the themes of genetics and somatic cells function. And at the Pasteur Institute in Paris, Len discovered that the galactoside-concentrating mechanism encoded by the permease gene in the LacZ operon is responsible for the autocatalytic increase in LacZ induction. These and other findings contributed to the discovery of the LacZ operon, which has laid the groundwork for modern molecular biology. At the NIH, Len discovered that adding pyruvate is sufficient for supporting clonal growth, and Lee worked on the characterization of enzymes in the histidine synthesis pathway in Salmonella. After moving to Stanford to join the burgeoning Genetics Department, Len and Lee studied mouse histocompatibility antigens to use as somatic cell markers in cultured cell lines, and Lee discovered a potential model for human Rh immunization during pregnancy. They then focused on the study of H-2 antigens and immunoglobulin allotypes, specifically a study of the genetics of the Ig heavy-chain (IgH) chromosome region. During this time, Len became increasingly aware of the need to characterize and isolate the different kinds of lymphocytes. Building on a machine constructed by a group at Los Alamos to sort cells by volume, Len added a fluorescence-detection system, and sought to measure the amount of fluorescence associated with individual cells and to sort cells according to this measure in addition to volume. He and his co-researchers titled the machine the Fluorescence-Activated Cell Sorter (FACS), perhaps the first ever biotech instrument, and used the machine to track the expression of genes encoding surface molecules that distinguish various kinds of lymphocytes and other cells. They made available to the scientific community a series of monoclonal reagents to mouse MHC and other cell-surface molecules. Since its first model, the FACS has been improved upon through the addition of cell surface markers and lasers, and through improvements in the software interface. One of the first applications of FACS in immunology further characterized the Ig isotype and allotype commitment of naive B cells and their memory (IgG+)-B-cell progeny. Len and Lee began to investigate B-cell subsets in the spleen, and continue to work on this today. They also study T-cell subsets in murine models and humans, and focus in particular on the loss of CD4 and CD8 in HIV, and the role of glutathione (GSH) in HIV.
Questions
Before EMA was introduced to indicate dead cells, how were dead cells identified? i.e. if dead cells caused significant false positives after fixation and permeabilization when using FACS, how did you previously compensate for that?
In what ways were you, as a scientist, affected by the McCarthyism that you so protested in the '50s?
By the way, so sorry about the delayed post!
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