Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function <em>In Vitro</em>

Abstract

Granulocytes play a key role in the body's innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry

“…Recent advances in instrument design and reagent development have enabled rapid progression in available measurement techniques in the field of flow cytometry. Specifically, image‐based flow cytometry adds a considerable amount of additional analysis capability that is not realized in traditional flow cytometry (McFarlin et al., ; McFarlin et al., ; Venable et al., ; Henning et al., ). In the area of reagent design, new products have been developed that allow exponential amplification of intracellular mRNA signals.…”