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Threshold responses in production of reactive oxygen metabolites in individual neutrophils detected by flow cytometry and microfluorimetry.

ABSTRACT: 1. The fluorescent compound 2',7'-dichlorofluorescein was used as an indicator of intracellular H2O2 production by neutrophils in order to compare the response of the cell population with that observed with individual cells determined by flow cytometry and quantitative fluorescence microscopy. 2. 2',7'-Dichlorofluorescein diacetate was deacetylated by intracellular esterases to form reduced 2',7'-dichlorofluorescein. The polar non-fluorescent intermediate remained trapped within both intracellular granules and the cytoplasm. Reduced dichlorofluorescein was oxidized by H2O2, a product of the oxidative burst, to yield the highly fluorescent product dichlorofluorescein. 3. A population of neutrophils stimulated by suboptimal concentrations of fMet-Leu-Phe (N-formylmethionyl-leucyl-phenylalanine) or phorbol ester (phorbol 12-myristate 13-acetate) resulted in an oxidation of 45-50% of the cellular dichlorofluorescein (non-fluorescent) to oxidized dichlorofluorescein within 30 min. Subcellular fractionation showed that, although dichlorofluorescein (non-fluorescent) occurred both in the cytoplasm and the granules, oxidation of dichlorofluorescein (non-fluorescent) occurred predominantly in the granules of stimulated neutrophils. 4. Flow cytometry showed that unstimulated cells consisted of a single population of cells with low cellular fluorescence. Activation of neutrophils (to produce reactive oxygen metabolites) resulted in the appearance of a second population of cells, with high fluorescence. The number of cells in this new population increased with time. fMet-Leu-Phe (0.1 microM) or phorbol ester (1 ng/ml) activated 45% of the cells within 8 min and 42% within 30 min respectively. 5. Analysis of individual cells by quantitative fluorescence microscopy demonstrated that, in the presence of a suboptimal concentration of stimulus, cells either failed to respond or were activated after different time delays, 4-120 s (39 +/- 18.4 s) by fMet-Leu-Phe or 12-200 s (59 +/- 17.4 s) by phorbol ester. Furthermore the oxidative bursts were of different magnitudes. 6. It is concluded that, in order for an individual cell to cross the activation threshold for the 'end response', a critical concentration of stimulus together with the necessary changes in intracellular signals are required.

SUBMITTER: Patel AK

PROVIDER: S-EPMC1148515 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Project description:Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus facilitating progress in quantitative studies of lymphocyte responses.

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Threshold responses in production of reactive oxygen metabolites in individual neutrophils detected by flow cytometry and microfluorimetry.

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