Flow cytometry: an introduction.

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells.

Dye dilution proliferation assay: application of the DDPA to identify tumor-specific T cell precursor frequencies in clinical trials.

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8(+) cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8(+) lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods.

A flow cytometric assay for quantitation of rare antigen-specific T cells: using cell-tracking dyes to calculate precursor frequencies for proliferation.

Cell-tracking dyes stain cells with bright fluorescence which is partitioned between daughter cells after each cell division, so that the daughter cells have closely half the intensity of the parent cells from which they were derived. Therefore, the intensity of a cell, relative to its intensity at the time of staining, provides information about how many divisions have occurred. Knowing the number of division cycles that have occurred, one can calculate the number of cells in the original population (before culture) that were going to go on to proliferate.

Decreased physiologic variability as a generalized response to human endotoxemia.

Objective: To test the effect in normal human volunteers of transient systemic inflammation on the variability in time-series behaviors of widely divergent physiologic measures of the human inflammatory response.

Design: Prospective study of human volunteers who were tested on 2 consecutive days, a control day and a treatment day. Each participant served as his or her own control.

Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cells.

The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation.

Flow cytometry: an introduction.

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells.

Multiparameter precursor analysis of T-cell responses to antigen.

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells.

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163.

CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo.

Insulin increases neutrophil count and phagocytic capacity after cardiac surgery.

Unlabelled: We previously reported that a continuous insulin infusion improves neutrophil phagocytic function after cardiac surgery in diabetic patients. These data suggested that hyperglycemia impairs neutrophil function, and because nondiabetic patients also experience hyperglycemia during cardiac surgery, we hypothesized that a continuous insulin infusion would improve glucose control and neutrophil function in nondiabetic cardiac surgical patients. Patients were randomized to receive either no insulin (Control group) or a continuous insulin infusion (Insulin group), with glucose measurements every 10 min during cardiopulmonary bypass (CPB).