Cytometry Part A最新文献

Due to the lack of an efficient in vitro spermatogenesis system, studies on mammalian spermatogenesis require the isolation of specific germ cell populations for further analyses. BSA gradient and elutriation have been used for several decades to purify testicular germ cells; more recently, flow cytometric cell sorting has become popular. Although each method has its advantages and disadvantages and is used depending on the purpose of the experiment, reliance on flow cytometric cell sorting is expected to be more prevalent because fewer cells can be managed. However, the currently used flow cytometric cell sorting method for testicular germ cells relies on karyotypic differences via DNA staining. Thus, it remains challenging to separate post-meiotic haploid cells (spermatids) according to their differentiation stage despite significant variations in morphology and chromatin state. In this study, we developed a method for finely separating testicular germ cells using VC mice carrying fluorescently tagged histones. This method enables the separation of spermatogonia, spermatocytes, and spermatids based on the intensity of histone fluorescence and cell size. Combined with a DNA staining dye, this method separates spermatids after elongation according to each spermiogenic stage. Although the necessity for a specific transgenic mouse line is less versatile, this method is expected to be helpful for the isolation of testicular germ cell populations because it is highly reproducible and independent of complex cell sorter settings and staining conditions.

Rodríguez-Martínez EA, Rivas CU, Ayala ME, Blanco-Rodríguez R, Juarez N, Hernandez-Vargas EA, et al. A new computational approach, based on images trajectories, to identify the subjacent heterogeneity of sperm to the effects of ketanserin. Cytometry. 2023; 103(8): 655–663. https://doi.org/10.1002/cyto.a.24732

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Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells—we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients—were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

Mesenchymal stem cells (MSCs) being injected into the body can stimulate or decelerate carcinogenesis. Here, the direction of influence of human placenta-derived MSCs (P-MSCs) on the Lewis lung carcinoma (LLC) tumor development and metastatic potential is investigated in C57BL/6 mice depending on the injection method. After intramuscular co-inoculation of LLC and P-MSCs (LLC + P-MSCs), the growth of primary tumor and angiogenesis are slowed down compared to the control LLC on the 15th day. This is explained by the fact of a decrease in the secretion of proangiogenic factors during in vitro co-cultivation of an equal amount of LLC and P-MSCs. When P-MSCs are intravenously (i.v.) injected in the mice with developing LLC (LLC + P-MSCs(i.v.)), the tumor growth and angiogenesis are stimulated on the 15th day. A highly activated secretion of proangiogenic factors by P-MSCs in a similar in vitro model can explain this. In both the models compared to the control on the 23rd day, there is no significant difference in the tumor growth, while angiogenesis remains correspondingly decelerated or stimulated. However, in both the models, the total volume and number of lung metastases constantly increase compared to the control: it is mainly due to small-size metastases for LLC + P-MSCs(i.v.) and larger ones for LLC + P-MSCs. The increase in the rate of LLC cell dissemination after the injection of P-MSCs is explained by the disordered polyploidy and chromosomal instability, leading to an increase in migration and invasion of cancer cells. After LLC + P-MSCs co-inoculation, the tumor cell karyotype has the most complex and heterogeneous chromosomal structure. These findings indicate a bidirectional effect of P-MSCs on the growth of LLC in the early periods after injection, depending on the injection method, and, correspondingly, the number of contacting cells. However, regardless of the injection method, P-MSCs are shown to increase LLC aggressiveness related to cancer-associated angiogenesis and metastasis activation in the long term.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a diagnostic sample into several tubes, restricting the number of cells and the complexity of immunophenotypes that can be analyzed. Full spectrum flow cytometers overcome this limitation by enabling the simultaneous use of up to 40 fluorescent markers. Here, we used this approach to develop a good laboratory practice-conform single-tube 19-color MRD detection assay that complies with recommendations of the European LeukemiaNet Flow-MRD Working Party. We based our assay on clinically-validated antibody clones and evaluated its performance on an IVD-certified full spectrum flow cytometer. We measured MRD and normal bone marrow samples and compared the MRD data to a widely used reference MRD-MFC panel generating highly concordant results. Using our newly developed single-tube panel, we established reference values in healthy bone marrow for 28 consensus leukemia-associated immunophenotypes and introduced a semi-automated dimensionality-reduction, clustering and cell type identification approach that aids the unbiased detection of aberrant cells. In summary, we provide a comprehensive full spectrum MRD-MFC workflow with the potential for rapid implementation for routine diagnostics due to reduced cell requirements and ease of data analysis with increased reproducibility in comparison to conventional FlowMRD routines.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Liver macrophages are critical components of systemic immune system defense mechanisms. F4/80^high Kupffer cells (KCs) are the predominant liver-resident macrophages and the first immune cells to contact pathogens entering the liver. F4/80^low monocyte-derived macrophages (MoMφs) are essential macrophages that modulate liver immune functions. Here we report a novel method of identifying subpopulations of these two populations using traditional flow cytometry and examine each subpopulation for its putative roles in the pathogenesis of an experimental non-alcoholic steatohepatitis model. Using male C57BL/6 mice, we isolated and analyzed liver non-parenchymal cells by flow cytometry. We identified F4/80^high and F4/80^low macrophage populations and characterized subpopulations using uniform manifold approximation and projection. We identified three subpopulations in F4/80^high macrophages: CD163(+) KCs, CD163(-) KCs, and liver capsular macrophages. CD163(+) KCs had higher phagocytic and bactericidal activities and more complex cellular structures than CD163(-) KCs. We also identified four subpopulations of F4/80^low MoMφs based on Ly6C and MHC class II expression: infiltrating monocytes, pro-inflammatory MoMφs, Ly6C(-) monocytes, and conventional dendritic cells. CCR2 knock-out mice expressed lower levels of these monocyte-derived cells, and the count varied by subpopulation. In high-fat- and cholesterol-diet-fed mice, only one subpopulation, pro-inflammatory MoMφs, significantly increased in count. This indicates that changes to this subpopulation is the first step in the progression to non-alcoholic steatohepatitis. The community can use our novel subpopulation and gating strategy to better understand complex immunological mechanisms in various liver disorders through detailed analysis of these subpopulations.