Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward-and sidescattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second. ©2013 Optical Society of America

The ability of the central nervous system to synthesize steroid hormones has wide-ranging implications for physiology and pathology. Among the proposed roles of neurosteroids is the regulation of the LH surge. This involvement in the estrogen-positive feedback demonstrates the integration of peripheral steroids with neurosteroids. Within the female hypothalamus, estradiol from developing follicles stimulates progesterone synthesis in astrocytes, which activate neural circuits regulating gonadotropin (GnRH) neurons. Estradiol acts at membrane estrogen receptor-α to activate cellular signaling that results in the release of inositol trisphosphate-sensitive calcium stores that are sufficient to induce neuroprogesterone synthesis. The purpose of the present studies was to characterize the estradiol-induced signaling leading to activation of steroid acute regulatory protein (StAR) and transporter protein (TSPO), which mediate the rate-limiting step in steroidogenesis, ie, the transport of cholesterol into the mitochondrion. Treatment of primary cultures of adult female rat hypothalamic astrocytes with estradiol induced a cascade of phosphorylation that resulted in the activation of a calcium-dependent adenylyl cyclase, AC1, elevation of cAMP, and activation of both StAR and TSPO. Blocking protein kinase A activation with H-89 abrogated the estradiol-induced neuroprogesterone synthesis. Thus, together with previous results, these experiments completed the characterization of how estradiol action at the membrane leads to the augmentation of neuroprogesterone synthesis through increasing cAMP, activation of protein kinase A, and the phosphorylation of TSPO and StAR in hypothalamic astrocytes.

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