Flow cytometry (FCM), as an important method for the diagnosis of acute myeloid leukemia (AML), has been widely used in myelodysplastic syndrome (MDS) in recent years. FCM can effectively identify and characterize abnormal hematopoietic cells through immunophenotyping, particularly in the diagnosis of MDS with insignificant morphological abnormalities. Additionally, FCM can monitor disease progression and minimal residual disease (MRD) by detecting phenotypic changes and evaluate treatment efficacy. This article reviews the latest research progress of FCM in the immunophenotyping, prognosis and efficacy evaluation of MDS.
Flow Cytometry ; Myelodysplastic Syndromes/therapy* ; Humans ; Leukemia, Myeloid, Acute/diagnosis* ; Immunophenotyping/methods* ; Prognosis

OBJECTIVE: To explore the influence of oligospermia (OS) on the detection of sperm DNA fragmentation index (DFI) by fluorescence method based on artificial intelligence (AI) recognition and flow cytometry-based sperm chromatin structure assay (SCSA). METHODS: We collected semen samples from 201 males, including 50 azoospermia (AS) patients as negative controls, 90 OS patients (sperm concentration >0×10⁶/ml and <15×10⁶/ml), and 61 normal men (sperm concentration ≥15×10⁶/ml). Then we subdivided the OS patients into a mild OS (sperm concentration ≥10×10⁶/ml and <15×10⁶/ml), a moderate OS (sperm concentration ≥5×10⁶/ml and <10×10⁶/ml) and a severe/extremely severe OS group (sperm concentration >0×10⁶/ml and <5×10⁶/ml), with 30 cases in each group, and compared the results of DFI detection between the AI fluorescence method and traditional flow cytometry. RESULTS: The DFI value detected by AI fluorescence method showed statistically significant difference from that detected by flow cytometry in the AS, moderate OS and severe/extremely severe OS groups (P<0.01), the former even lower than the latter, but not in the normal control and the mild OS groups (P > 0.05). In the AS group, a dramatically lower rate of non-0 results was achieved by AI fluorescence method than by flow cytometry (8% vs 100%, P<0.01). The DFI values detected by AI fluorescence method exhibited a good linear correlation to those obtained by flow cytometry in the normal control and mild OS groups (R2 = 0.7470; R2 = 0.7180), but a poor linear correlation in the OS full-sample, moderate OS and severe/extremely severe OS groups (R2 = 0.3092; R2 = 0.3558; R2 = 0.2147). CONCLUSION The AI fluorescence method has a higher specificity and is more suitable than flow cytometry for detection of sperm DFI in OS patients. The DFI values obtained by the two methods are consistent with sperm concentration ≥10×10⁶/ml, but the accuracy of the results of detection may be affected with sperm concentration >0×10⁶/ml and <10×10⁶/ml.
Humans ; Male ; Flow Cytometry/methods* ; Oligospermia/genetics* ; Artificial Intelligence ; Spermatozoa ; Adult ; DNA Fragmentation ; Case-Control Studies ; Fluorescence

Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1^CreERT2; R26^GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6^f/f; CX3CR1^CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnfα promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnfα transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.
Animals ; Mice ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Tooth Movement Techniques/methods* ; Activating Transcription Factor 6/metabolism* ; Bone Remodeling ; Flow Cytometry ; Blotting, Western

This study aims to establish a method for counting the viral particles in adenovirus vector-based vaccines. Nano-flow cytometry was employed to analyze the viral particles in adenovirus-based vector vaccines at the single-particle level. Monodisperse silica nanoparticles with a refractive index close to that of the virus were selected as the particle size standard to calculate the viral particle size, which was then compared with the results obtained from transmission electron microscopy (TEM) to determine the gating strategy. Subsequently, a particle count standard was employed to calculate the viral particle concentration. The established method demonstrated good linearity, accuracy, precision, and specificity. The results of determined viral particle concentration showed a good correlation with the infectious titer. Compared with the conventional OD₂₆₀ method, nano-flow cytometry can directly measure the viral particle concentration and indicate whether the sample has been disassembled according to changes in viral particle concentration and size, thus more accurately reflecting the actual infectious potency of the sample. The novel quantification method established in this study is capable of indicating the efficacy of adenovirus vector-based vaccines and provides effective technical support for the quality control of such products.
Adenoviridae/genetics* ; Genetic Vectors ; Flow Cytometry/methods* ; Virion/isolation & purification* ; Particle Size ; Nanoparticles ; Viral Vaccines

Actinomycetes are important producers of high-value natural products, and the engineering of actinomycetes to enhance the biosynthesis of target natural products has long been a hot research topic in the scientific community. However, non-rational engineering methods suffer from low beneficial mutation rates, which limit the efficiency of mutant screening. The integration of high-throughput screening (HTS) technologies can effectively enhance the screening efficiency of elite mutants and significantly shorten the cycle of actinomycete strain engineering. This review comprehensively discusses various HTS technologies suitable for the engineering of actinomycete strains and compares them in terms of application scenarios, advantages, and disadvantages. HTS technologies include microplate-based screening, antimicrobial activity screening, antibiotic resistance screening, fluorescence-activated cell sorting (FACS), and fluorescence-activated droplet sorting (FADS). Additionally, this review summarizes the applications of these technologies in assisting actinomycete strain engineering and enhancing the yields of target compounds. The development and application of HTS technologies have not only facilitated the exploration of natural product resources in actinomycetes but also provided strong support for the rapid and efficient construction of high-performance engineered actinomycete strains.
Actinobacteria/metabolism* ; High-Throughput Screening Assays/methods* ; Genetic Engineering/methods* ; Biological Products/metabolism* ; Flow Cytometry ; Metabolic Engineering/methods*

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4⁺ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4⁺ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Cattle ; Animals ; Cytokines ; BCG Vaccine/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-2 ; Flow Cytometry/methods* ; Chemokine CXCL10/metabolism* ; Leukocytes, Mononuclear ; CD4-Positive T-Lymphocytes/metabolism* ; Tuberculosis ; Antibodies, Monoclonal/metabolism*

Flow cytometry is a multi-parameter, rapid and efficient method for qualitative analysis and quantitative determination of various fluorescently labeled particles in liquid flow. Flow cytometry has been applied in multiple disciplines such as immunology, virology, molecular biology, cancer biology and infectious disease monitoring. However, the application of flow cytometry in plant research is hampered due to the special composition and structure of plant tissues and cells, such as cell walls and secondary metabolites. In this paper, the development, composition and classification of flow cytometry were introduced. Subsequently, the application, research progress and application limitations of flow cytometry in plant field were discussed. At last, the development trend of flow cytometry in plant research was prospected, which provides new perspectives for broadening the potential application scope of plant flow cytometry.
Flow Cytometry/methods* ; Plants ; Fluorescent Dyes

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.
Mice ; Animals ; Astrocytes/metabolism* ; Microglia/metabolism* ; Cell Count ; Flow Cytometry/methods* ; Cell Proliferation ; Cells, Cultured

The detection of immune cell subsets plays a very important role in the clinical diagnosis and treatment of various benign and malignant diseases and health management. In order to better carry out in-depth research on different functional immune cell subsets, establish reference intervals for clonality related indicators, establish special reference intervals for immune aging, individualized dynamic monitoring and treatment recovery, and discover the clinical significance of immune cells other than lymphocytes, it is urgent to analyze the peripheral blood immune cell subsets in a refined way. Multiparameter flow cytometry is an important technical method to detect immune cell subsets and evaluate immune function. In order to standardize the refined detection methods and protocols of peripheral blood immune cell subsets by flow cytometry, and further promote its application in clinical diagnosis and treatment of diseases and health management, Laboratory Medicine Committee of Chinese Association of Integrative Medicine (LMC-CAIM) organized experts to formulate this expert consensus.
Humans ; Consensus ; East Asian People ; Flow Cytometry/methods* ; Immune System/cytology*

Objective To examine the antiplatelet effect of ticagrelor by microfluidic chip and flow cytometry under shear stress in vitro. Methods Microfluidic chip was used to examine the effect of ticagrelor on platelet aggregation at the shear rates of 300/s and 1500/s.We adopted the surface coverage of platelet aggregation to calculate the half inhibition rate of ticagrelor.The inhibitory effect of ticagrelor on ADP-induced platelet aggregation was verified by optical turbidimetry.Microfluidic chip was used to construct an in vitro vascular stenosis model,with which the platelet reactivity under high shear rate was determined.Furthermore,the effect of ticagrelor on the expression of fibrinogen receptor (PAC-1) and P-selectin (CD62P) on platelet membrane activated by high shear rate was analyzed by flow cytometry. Results At the shear rates of 300/s and 1500/s,ticagrelor inhibited platelet aggregation in a concentration-dependent manner,and the inhibition at 300/s was stronger than that at 1500/s (both P<0.001).Ticagrelor at a concentration ≥4 μmol/L almost completely inhibited platelet aggregation.The inhibition of ADP-induced platelet aggregation by ticagrelor was similar to the results under flow conditions and also in a concentration-dependent manner.Ticagrelor inhibited the expression of PAC-1 and CD62P. Conclusion We employed microfluidic chip to analyze platelet aggregation and flow cytometry to detect platelet activation,which can reveal the responses of different patients to ticagrelor.
Humans ; Ticagrelor/pharmacology* ; Platelet Aggregation Inhibitors/pharmacology* ; Flow Cytometry/methods* ; Microfluidics ; Platelet Aggregation