A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution.

Labeling nonquiescent cells with carboxyfluorescein succinimidyl ester (CFSE)-like dyes gives rise to a population width exceeding the threshold for resolving division peaks by flow cytometry. Width is a function of biological heterogeneity plus extrinsic and intrinsic error sources associated with the measurement process. Optimal cytometer performance minimizes extrinsic error, but reducing intrinsic error to the point of facilitating peak resolution requires careful fluorochrome selection and fluorescent cell sorting. In this study, we labeled the Jurkat and A549 cell lines with CFSE, CellTraceViolet (CTV), and eFluor 670 proliferation dye (EPD) to test if we could resolve division peaks in culture after reducing the labeled input widths by cell sorting. Reanalysis of the sorted populations to ascertain the level of reduction achieved always led to widths exceeding the gated limits due to the contribution of errors. Measuring detector-specific extrinsic error by sorting uniform fluorescent particles with similar spectral properties to the tracking dyes allowed us to determine the intrinsic error for each dye and cell type using a simple mathematical approach. We found that cell intrinsic error ultimately dictated whether we could resolve division peaks, and that as this increased, the required sort gate width to resolve any division peaks decreased to the point whereby issues with yield made A549 unsuitable for this approach. Finally, attempts to improve yields by setting two concurrent sort gates on the fluorescence distribution enriched for cells in different stages of the cell cycle that had nonequivalent proliferative properties in culture and thus should be practiced with caution.

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival.

Label-free cell cycle analysis for high-throughput imaging flow cytometry.

Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types.

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.

Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to combine mitochondrial Ca(2+) measurements with nuclear translocation. In conclusion, this method enables the high throughput study of spatiotemporal patterns of Ca2(+) signals in T cells responding to different stimuli.

Short-term exposure of nontumorigenic human bronchial epithelial cells to carcinogenic chromium(VI) compromises their respiratory capacity and alters their bioenergetic signature.

Previous studies on the impact of hexavalent chromium [Cr(VI)] on mammalian cell energetics revealed alterations suggestive of a shift to a more fermentative metabolism. Aiming at a more defined understanding of the metabolic effects of Cr(VI) and of their molecular basis, we assessed the impact of a mild Cr(VI) exposure on critical bioenergetic parameters (lactate production, oxygen consumption and intracellular ATP levels). Cells derived from normal human bronchial epithelium (BEAS-2B cell line), the main in vivo target of Cr(VI) carcinogenicity, were subjected for 48 h to 1 µM Cr(VI). We could confirm a shift to a more fermentative metabolism, resulting from the simultaneous inhibition of respiration and stimulation of glycolysis. This shift was accompanied by a decrease in the protein levels of the catalytic subunit (subunit ß) of the mitochondrial H(+)-ATP synthase (ß-F1-ATPase) and a concomitant marked increase in those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The corresponding alteration in the ß-F1-ATPase/GAPDH protein ratio (viewed as a bioenergetic signature) upon Cr(VI) exposure was in agreement with the observed attenuation of cellular respiration and enhancement of glycolytic flux. Altogether, these results constitute a novel finding in terms of the molecular mechanisms of Cr(VI) effects.