The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of ...pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX® system and RAPID-B® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX® system and RAPID-B® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B® flow cytometer and USDA-FSIS reference method. The RAPID-B® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B® flow cytometer based on whole cell detection generated similar results as BAX® system therefore the RAPID-B® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products.
•We compared the non-O157 STEC detection sensitivity between BAX® system and RAPID-B® flow cytometer.•The RAPID-B® flow cytometer exhibited similar results as BAX® system.•The RAPID-B® flow cytometer is a rapid detection method for non-O157 STEC in beef products.
Purpose
Liposomes have been developed as versatile nanocarriers for various pharmacological agents. The effect of surface charges on the cellular uptake of the liposomes has been studied by various ...methods using mainly fixed cells with inevitable limitations. Live cell imaging has been proposed as an alternative methods to overcome the limitations of the fixed cell-based analysis. In this study, we aimed to investigate the effects of surface charges on cellular association and internalization of the liposomes using live cell imaging.
Methods
We studied the cellular association and internalization of liposomes with different surface charge using laser scanning confocal microscopy (LSCM) equipped with live cell chamber system. Flow cytometry was also carried out using flow cytometer (FACS) for comparison.
Results
All of the cationic, neutral and anionic liposomes showed time-dependent cellular uptake through specific endocytic pathways. In glioblastoma U87MG cells, the cationic and anionic liposomes were mainly taken up
via
macropinocytosis, while the neutral liposomes mainly
via
caveolae-mediated endocytosis. In fibroblast NIH/3T3 cells, all of the three liposomes entered into the cell
via
clathrin-mediated endocytosis.
Conclusions
This study provides a better understanding on the cellular uptake mechanisms of the liposomes, which could contribute significantly to development of liposome-based drug delivery systems.
The strategy of identification for M1 and M2 macrophages both in vivo and in vitro would help to predict the health condition of the individual. Here, we introduced a solution to this problem with ...the advantage of both the phagocytic nature of macrophages and the scattering effect of gold nanorods (GNRs). The internalized GNRs, relating to their extent of intake, caused a conspicuous scattering profile at the red channel in flow cytometry, overruling the contribution of the cellular side scatters. This internalization is solely governed by the surface chemistry of GNRs. The PAH-GNRs showed maximum intake potency followed by Cit-, PSS-, and PEG-GNRs. On a substantial note, PAH-GNRs lead to differential uptake between M1 and M2 cells, with three times higher intake in M2 cells over M1. This is the first report of employing the scattering of unlabeled GNRs to discriminate M1 and M2 cell types using a flow cytometer.
Recent study proves that the combination of loop mediated isothermal nucleic acid amplification (LAMP) with one-step strand displacement (OSD) is of great help to improve the sequence specificity ...during genetic detection. However, because OSD is incapable of signal amplification, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. With the purpose to overcome this challenge, herein a more advanced and practical sensing principle is developed with the OSD replaced by an amplifiable nucleic acid circuit, hybridization chain reaction (HCR). The very contagious norovirus (NoV) was employed as the model target. Compared with LAMP-OSD, the LAMP-HCR can detect as few as 30 copies of NoV gene in 2% fecal samples with significantly enlarged signal change and signal-to-background ratio. Therefore, more reliable detection is achieved. Moreover, due to the high compatibility of HCR, the final LAMP-HCR products can be flexibly transduced into different types of readouts, including fluorescence, flow cytometer (FCM) and even a personal glucose meter (PGM). This further enlarges the operating environments for the detection from hospital labs, bedsides, to potential off-the-shelf devices in local pharmacies. Especially when using FCM or PGM, with the assistance of magnetic beads (MBs), the detection shows even higher tolerance capability to complicated biological matrices.
A versatile molecular diagnostic method is developed for sensitive norovirus (NoV) gene detection by coupling the advantages of loop-mediated isothermal amplification (LAMP) with enzyme-free signal amplification hybridization chain reaction (HCR) for robust signal readout via flow cytometer (FCM) and personal glucometer (PGM). Display omitted
•Highly sensitive and selective detection method for norovirus gene.•Sensitivity down to 30 gene copies in 2% fecal samples.•Significantly enlarged signal change and signal-to-background ratio.•Higher tolerance capability to complicated biological matrices.•Exhibiting great potential to be used in the diagnosis of gastroenteritis in hospital or at home.
The objective of the current research work was to fabricate a fosfestrol (FST)-loaded self-nanoemulsifying drug delivery system (SNEDDS) to escalate the oral solubility and bioavailability and ...thereby the effectiveness of FST against prostate cancer.
3
full factorial design was employed, and the effect of lipid and surfactant mixtures on percentage transmittance, time required for self-emulsification, and drug release were studied. The optimized solid FST-loaded SNEDDS (FSTNE) was characterized for in vitro anticancer activity and Caco-2 cell permeability, and in vivo pharmacokinetic parameters.
Using different ratios of surfactant and co-surfactant (Km) a pseudo ternary phase diagram was constructed. Thirteen liquid nano emulsion formulations (LNE-1 to LNE-13) were formulated at Km = 3:1. LNE-9 exhibited a higher % transmittance (99.25 ± 1.82 %) and a lower self-emulsification time (24 ± 0.32 s). No incompatibility was observed in FT-IR analysis. Within 20 min the solidified FST loaded LNE-9 (FSTNE) formulation showed almost complete drug release (98.20 ± 1.30 %) when compared to marketed formulation (40.36 ± 2.8 %), and pure FST (32 ± 3.3 %) in 0.1 N HCl. In pH 6.8 phosphate buffer, the release profiles are found moderately higher than in 0.1 N HCl. FSTNE significantly (P < 0.001) inhibited the PC-3 prostate cell proliferation and also caused apoptosis (P < 0.001) compared to FST. The in vitro Caco-2 cell permeability study results revealed 4.68-fold higher cell permeability of FSTNE than FST. Remarkably, 4.5-fold rise in bioavailability was observed after oral administration of FSTNE than plain FST.
FSTNE remarkably enhanced the in vitro anticancer activity and Caco-2 cell permeability, and in vivo bioavailability of FST. Thus, FST-SNEDDS could be utilized as a potential carrier for effective oral treatment of prostate cancer.
Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally ...attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix.
We develop a high-throughput technique to relate positions of individual cells to their three-dimensional (3D) imaging features with single-cell resolution. The technique is particularly suitable for ...nonadherent cells where existing spatial biology methodologies relating cell properties to their positions in a solid tissue do not apply. Our design consists of two parts, as follows: recording 3D cell images at high throughput (500 to 1,000 cells/s) using a custom 3D imaging flow cytometer (3D-IFC) and dispensing cells in a first-in-first-out (FIFO) manner using a robotic cell placement platform (CPP). To prevent errors due to violations of the FIFO principle, we invented a method that uses marker beads and DNA sequencing software to detect errors. Experiments with human cancer cell lines demonstrate the feasibility of mapping 3D side scattering and fluorescent images, as well as two-dimensional (2D) transmission images of cells to their locations on the membrane filter for around 100,000 cells in less than 10 min. While the current work uses our specially designed 3D imaging flow cytometer to produce 3D cell images, our methodology can support other imaging modalities. The technology and method form a bridge between single-cell image analysis and single-cell molecular analysis.
This article presents the development and testing of a low‐cost (<$60), portable, electrical impedance‐based microflow cytometer for single‐cell analysis under a controlled oxygen microenvironment. ...The system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. Impedance values of sickle blood samples exhibit remarkable deviations from the common reference line obtained from two normal blood samples. Such deviation is quantified by a conformity score, which allows for the measurement of intrapatient and interpatient variations of sickle cell disease. A low conformity score under oxygenated conditions or drastically different conformity scores between oxygenated and deoxygenated conditions can be used to differentiate a sickle blood sample from normal. Furthermore, an equivalent circuit model of a suspended biological cell is used to interpret the electrical impedance of single flowing RBCs. In response to hypoxia treatment, all samples, regardless of disease state, exhibit significant changes in at least one single‐cell electrical property, that is, cytoplasmic resistance and membrane capacitance. The overall response to hypoxia is less in normal cells than those affected by sickle cell disease, where the change in membrane capacitance varies from −23% to seven times as compared with −17% in normal cells. The results reported in this article suggest that the developed method of testing demonstrates the potential application for a low‐cost screening technique for sickle cell disease and other diseases in the field and low‐resource settings. The developed system and methodology can be extended to analyze cellular response to hypoxia in other cell types.
A portable electrical impedance‐based microflow cytometer was developed to measure cellular response to hypoxia. The conformity score assessed from the impedance signals of red blood cells showed substantial intra‐ and interpatient variability in sickle cell disease. This development brings affordability to the diagnosis and monitoring of sickle cell disease in the point of care and resource limited settings.
PDF
