Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a ...measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.
Flow cytometry is a laser-based technology that rapidly detects and analyzes the chemical and physical characteristics of single cells or particles and is already well established in environmental ...and toxicological studies for microalgae and bacterial quantification. This study introduces an imaging Flow Cytometer (FC) system, designed specifically for the enhanced analysis of microalgae biomass populations and aggregate groups through Artificial Intelligence (AI) integration. The FC incorporates a single flow line, critical hardware components, and a trifurcated software setup. The system employs a multi-step process for counting algae units and an Artificial Neural Network (ANN) for classifying them in groups of two or four. To demonstrate its capabilities, the system was tested on its ability to capture, count, and categorize algal units, specifically the Desmodesmus sp. morphotype with high accuracy. Furthermore, the FC's capabilities were contrasted with traditional counting methods, validating its enhanced precision and efficiency against a hematocytometer. With its capability to provide rapid, accurate, and high-throughput analyses, this innovative FC paves the way for a revolutionary approach to cellular research.
•FACS is a useful tool to separate soil microbes of different sizes.•Different cell size classes harbor distinct bacteria communities.•Bacterial core community varies according to size classes and ...soil type.•Bacterial functional similarities of different size classes depend on soil type.
Cell size is a key morphological trait which is associated with microbial activity and nutrient acquisition. However, it is still unclear whether bacteria of different sizes have similar structural and functional properties. In this study, we sorted bacterial cells into five size classes (small, slightly small, medium, slightly large, large) using FACS and compared their structural and functional profiles in soils from deciduous and evergreen forests. The results showed that most (about 60%) of the bacterial cells fell under small or slightly small size classes. The five size classes harbored distinct bacterial communities in both types of forest soil. In spite of the lower relative abundance, slightly large and large bacterial cells had higher diversity compared to other size classes. Core communities of the five size classes in evergreen forest soil harbored more ubiquitous OTUs when compared with deciduous forest soil. Bacterial functional structures of the five size classes were significantly different in deciduous forest soil, while similar across size classes in evergreen forest soil. We conclude that cell size is an important factor that determines bacterial structure and function, and this relationship is depended on ecosystem or soil type. This study emphasizes cell size as a useful tool when assessing bacterial diversity and uncovers a direct relationship between microbial morphology and ecological traits.
Tumor markers play a significant role in early cancer diagnosis, evaluation of the extent of the disease, and monitoring of therapy response. In this study, we described the Pickering emulsion ...polymerization method to synthesize uniform magnetic/fluorescent microspheres. A Pickering-structure composed of a lot silica nanoparticle closely covered onto the quantum dot-encoded magnetic microbeads is designed and synthesized. The uniform magnetic/fluorescent microspheres were prepared using a microfluidic device and the performance of the microspheres synthesized by the instruments was evaluated by flow cytometry. To avoid fluorescence quenching and intrinsic toxicity, CdSe/ZnS core-shell quantum dot and Fe3O4 nanoparticle were successfully encapsulated into MFM microspheres using the microfluidic technology. Using this structure enables the facile realization of a theoretical 4 × 4 barcoding matrix combining two colors and four fluorescence intensity levels. Then, different optical codes were prepared by simple changing the emission wavelength and the intensity of the quantum dots. The resulting microsphere are combined with flow cytometer using two lasers for decoding of multiplex tumor markers. Moreover, the stability testing of microspheres demonstrated good performance for further application in detection of tumor markers as well. When applied for the high-throughput ultrasensitive detection of three tumor markers (CEA, CA125 and CA199) in a single sample, the detection limits of 0.027 ng/mL for CEA, 1.48 KU/L for CA125 and 1.09 KU/L for CA199 are achieved, which exhibit superior detection performance. Thus, Pickering-structure magnetic/fluorescent microspheres are promising for application in tumor markers.
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•One-step method integrates optical barcodes and magnetic nanoparticles into microspheres for simple and low-cost operation.•Microspheres synthesized by Pickering emulsion polymerization have a high specific surface area.•Silica nanoparticles as stabilizers not only enhance the stability of the microspheres, but also make the microspheres easier to modify, expanding the field of use of the microspheres.
Type-2 diabetes, characterized by hyperglycemia causing various symptoms of metabolic disorders in the heart, kidneys, and brain, has many underlying molecular mechanisms leading to functional ...insufficiency of these organs. We describe protocols wherein we have optimized conditions for maintenance of hyperglycemic H9c2 cell lines and design to assess the effect of a water-soluble vitamin, Trolox, on the apoptotic pathway. Primarily, the design provides researchers to analyze apoptosis by flow cytometry.
Preliminary studies have shown that sperm membrane from swine shows high sensitivity to cryopreservation process, causing a dramatic reduction in sperm quality. This has been attributed to the ...production of reactive oxygen species that cause lipid peroxidation in sperm membranes. The aim of the present study was to minimize the oxidative attack by adding different concentration of alpha-lipoic acid into the sperm liquid storage at 17ºC for 7 days. Freshly ejaculated boar semen was diluted with Beltsville Thawing Solution (BTS) and supplemented with 5 levels of alpha-lipoic acid (0.015, 0.02, 0.05, 0.1, 0.15 mmol/ml). The membrane integrity was evaluated at days 0, 1, 3, 5 and 7 of liquid preservation, using flow cytometer FACSCanto II (BD Biociencias) systems. The experiment indicate that supplementation of alpha-lipoic acid to the semen liquid storage extender improve sperm membrane.
Introduction: The main strategy to reduce the immunogenicity of transplanted grafts is to seek maximum compatibility betweenalloantigens in the donor-recipient pair. It is understood that the virtual ...crossmatch (VXM) can be a good tool in the evaluation of the donor / recipient pair and that a broader application of these protocols will improve the pre-transplant immunological risk assessment. The aim of this study was to correlate the physical flow crossmatches results, looking at different mean fluorescence intensity (MFI) values of the donor specific antibodies (DSA), and the probability of a positive crossmatch. We also aimed to validate this tool using a well standardized flow crossmatch protocol. Methods: We performed a total of 15,217 FCXM between 2015 and 2019. All were tested by the Halifax Flow Cytometer Crossmatch (FCXM) protocol, with cells from deceased donors and serum from renal recipients. For this analysis we selected only samples that had one or two DSA per locus (N = 1,081), when the MFI was above 1,000, and they were divided according to the allelic group recognized by the antibody (anti-HLA-A, B, C, DR or DQ) looking at the probability of a positive crossmatch with different MFI values of the DSAs. Combinations among them were alsoanalyzed in the same way (N=175). Results: In the presence of an exclusive DSA against the allelic groups A, B and DR, with an index MFI above 5,000, all the FCXM were positives. With exclusively antibodies against groups C or DQ, all cases with DSAsabove 15,000 MFIs were positives. With two or more DSAs anti A and/or B, and/or DR, when their MFIs sums exceeded 5,000,all FCXM results against B cells were positive. The presence of anti-Class I DSAs (A, B and A+B), regardless of the MFI value, was responsible for 71% (N=424 of 601) of the T cell positivity and 77% (N=460 of 661) in the B cell crossmatches. The presence of only anti-Class II DSAs (DR and DQ) accounted for 55% (N=168 of 303) of FCXM positivity in B cells. The overall mean of the MFI of the DSAs was higher in the FCXM positive group when compared to negative crossmatch. The group with sum of DSAs A (N=217) showed that when the sum was 4,000 MFI or higher, there was a 24 times higher probability for a positive B cell crossmatch when compared with lowers MFIs. Conclusions: Our results show a strong association between the DSA MFI and the FCXM result. The data here presented confirm the results of our previous studies, justifying the VXMstandard used by our center, proving to be a good tool to streamline the selection of transplant recipients and facilitate the sharing of organs from national donors.
As a ubiquitous contaminant in aquatic environments, diethyl phthalate (DEP) is a major threat to ecosystems because of its increasing utilization. However, the ecological responses to and toxicity ...mechanisms of DEP in aquatic organisms remain poorly understood. To address this environmental concern, we selected Chlorella vulgaris (C. vulgaris) as a model organism and investigated the toxicological effects of environmentally relevant DEP concentrations at the individual, physiological, biochemical, and molecular levels. Results showed that the incorporation of DEP significantly inhibited the growth of C. vulgaris, with inhibition rates ranging from 10.3 % to 83.47 %, and disrupted intracellular chloroplast structure at the individual level, while the decrease in photosynthetic pigments, with inhibition rates ranging from 8.95 % to 73.27 %, and the imbalance of redox homeostasis implied an adverse effect of DEP at the physio-biochemical level. Furthermore, DEP significantly reduced the metabolic activity of algal cells and negatively altered the cell membrane integrity and mitochondrial membrane potential. In addition, the apoptosis rate of algal cells presented a significant dose–effect relationship, which was mainly attributed to the fact that DEP pollutants regulated Ca2+ homeostasis and further increased the expression of Caspase-8, Caspase-9, and Caspase-3, which are associated with internal and external pathways. The gene transcriptional expression profile further revealed that DEP-mediated toxicity in C. vulgaris was mainly related to the destruction of the photosynthetic system, terpenoid backbone biosynthesis, and DNA replication. Overall, this study offers constructive understandings for a comprehensive assessment of the toxicity risks posed by DEP to C. vulgaris.
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•Exploring the potential toxic mechanisms of DEP on microalgae from multiple angles•DEP initiates the antioxidant system of microalgae by activating SOD and POD viability.•The apoptosis pathway (internal and external pathway) is discussed.•DEP interferes with photosynthesis-antenna proteins and DNA replication in microalgae.
Sperm DNA integrity is important for normal functions such as fertilization, implantation, pregnancy and fetal development. Sperm DNA fragmentation (SDF) is more common in infertile men and may be ...responsible for poor reproductive function. Although there are a number of tests available to measure SDF, the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐nick end labelling TUNEL) assay using flow cytometry is becoming more popular to measure the sperm DNA fragmentation. It is a direct test that measures both single‐ and double‐ DNA strand breaks. In this review, we describe the protocol, quality control and measurement of sperm DNA fragmentation using a benchtop flow cytometer. We also briefly discuss the factors that can affect the results, challenges and clinical implications of TUNEL in assessing male infertility.
Safety of potable reuse can be enhanced by improved water quality monitoring techniques for assessing water treatment processes. This study evaluated the efficacy of online bacterial counting for ...continuous monitoring of reverse osmosis (RO) membranes to remove bacteria using real-time bacteriological commercial counters and an on-site pilot-scale RO system. Prior to on-site assessments, the online bacterial counting was verified by comparing the measurement of fluorescent particles in water with flow cytometry. During a seven day pilot test of RO treatment at a water reclamation plant, online bacterial counts in RO permeate were monitored below 15 counts/mL; whereas the bacterial counts in RO feed water were approximately 2500 to 10,000 counts/mL. Removal rates of bacterial counts ranged from 2.6 to 3.1-log (average = 2.9-log) by continuously monitoring bacterial removal. This is greater than a 2-log reduction frequently determined using other water quality surrogates (i.e., electrical conductivity). Overall, the continuous monitoring of bacteria in RO feed and permeate can be implemented without the addition of chemicals to provide near real-time bacterial counts to measure their reduction after RO treatment. This can be developed for continuous performance monitoring of the RO process, providing greater assurance of microbial water quality after RO treatment.
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•Efficacy of online bacterial counting for monitoring RO system integrity was assessed.•Real-time bacteriological commercial counters were used for this study's assessment.•Online bacterial counting was verified by measuring surrogates.•Bacterial counts in RO feed and permeate were monitored online at the pilot scale.•A range of 2.6 to 3.1-log reduction in bacteria was determined by online monitoring.
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