In Vivo Studies and Flow Cytometric Investigation on Anticancer Potential of Selenium Nanoparticles Synthesized via Aqueous Extract of Clerodendron phlomidis

Nowadays,doctors and nutritionists recommend individuals incorporate selenium-rich foods such as nuts,cereals,and mushrooms into their regular diet to maintain fitness and overall health.Selenium nanoparticles(SeNPs)exhibit strong chemopreventive capabilities.The anticipations for SeNPs with enhanced and tunable bioactive activities have led to a keen interest in phytofabrication.In this study,the aqueous extract of Clerodendron phlomidis plant leaves was utilized for the synthesis of SeNPs.In traditional Indian medicine,this plant extract is recognized as a significant anti-diabetic agent.The flavonoids tetrahydroxylflavone,7-hydroxyflavanone,and 6,4’-dimethyl-7-acetoxy-scutellarein present in this plant leaf extract demonstrate excellent anticancer activity.These secondary metabolites exhibit the ability to reduce sodium selenite into SeNPs.At a concentration of 13μg/mL,the synthesized SeNPs effectively inhibited the proliferation of the HepG2 cell line.The results suggest that the SeNPs possess promising anti-cancer potential against liver cancer and can be considered as a therapeutic agent for liver cancer treatment.Additionally,the cell cycle arrest induced by SeNPs was further confirmed by the fluorescence-activated cell sorting(FACS)method,indicating that SeNPs could efficiently differentiate cancer cells from normal cells.Notably,it showed a significant improvement in diethylnitrosamine(DEN)-induced Swiss Wistar rat groups.This scientific investigation highlights the high anti-cancer potential of SeNPs,positioning them as a promising therapeutic agent for liver cancer treatment.

Flow-rSSO及PCR-SBT方法检测KIR基因有无的对比研究

目的研究流式磁珠序列特异性寡核苷酸探针(Flow-rSSO)杂交及测序分型(PCR-SBT)方法鉴定KIR基因有无的一致性。方法对131例汉族人群的DNA标本采用Flow-rSSO方法鉴定全部16种KIR基因的有无,并采用本实验室建立的PCR-SBT方法对14种功能性KIR基因进行高分辨水平基因检测;分析Flow-rSSO及PCR-SBT两种方法鉴定14种功能性KIR基因有无的一致性。对初检结果不一致的标本,采用同一厂家、不同批号的Flow-rSSO试剂盒进行复检,并采用序列特异性引物-PCR(PCR-SSP)方法进行检测。结果131例标本中有129例完全一致,2例不一致,占1.5%(2/131)。不一致的两例标本,1例标本3DL1基因、另1例标本2DS3及2DS5基因,其Flow-rSSO初检结果均为阴性,而PCR-SBT结果均为阳性。更换新的批号Flow-rSSO试剂盒复检,两例标本的结果均为阳性;采用PCR-SSP方法检测的结果亦为阳性。结论经Flow-rSSO试剂盒检测KIR基因有无,出现2例标本初检结果错误,提示检测KIR基因有无时,试剂的质控工作至关重要。

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Detection of cells by flow cytometry:Counting,imaging,and cell classification

The study of circulating cells in the blood stream is critical,as it covers many felds of biomed-icine,including immunology,cell biology,oncology,and reproductive medicine.In-viuo flowcytometry(IVFC)is a new tool to monitor and count cells in real time for long durations in theirnative biological environment.This review describes two main categories of IVFC,ie.,labeledand label-free IVFC.It focuses on label-free IVFC and introduces its technological developmentand related biological applications.Because cell recognition is the basis of flow cytometrycounting,this review also describes various methods for the classification of unlabeled cells,including the latest machine learning-based technologies.

A multichannel thermal bubble-actuated impedance flow cytometer with on-chip TIA based on CMOS-MEMS

Electrochemical impedance spectroscopy(EIS)flow cytometry offers the advantages of speed,affordability,and portability in cell analysis and cytometry applications.However,the integration challenges of microfluidic and EIS read-out circuits hinder the downsizing of cytometry devices.To address this,we developed a thermal-bubble-driven impedance flow cytometric application-specific integrated circuit(ASIC).The thermal-bubble micropump avoids external piping and equipment,enabling high-throughput designs.With a total of 36 cell counting channels,each measuring 884×220μm^(2),the chip significantly enhances the throughput of flow cytometers.Each cell counting channel incorporates a differential trans-impedance amplifier(TIA)to amplify weak biosensing signals.By eliminating the parasitic parameters created at the complementary metal-oxidesemiconductor transistor(CMOS)-micro-electromechanical systems(MEMS)interface,the counting accuracy can be increased.The on-chip TIA can adjust feedback resistance from 5 to 60 kΩto accommodate solutions with different impedances.The chip effectively classifies particles of varying sizes,demonstrated by the average peak voltages of 0.0529 and 0.4510 mV for 7 and 14μm polystyrene beads,respectively.Moreover,the counting accuracies of the chip for polystyrene beads and MSTO-211H cells are both greater than 97.6%.The chip exhibits potential for impedance flow cytometer at low cost,high-throughput,and miniaturization for the application of point-of-care diagnostics.

Performance Evaluation of Combined Detection of Serum CEA,CYFRA21-1,CA125,and NSE in Patients with Lung Cancer by Fluorescence Flow Cytometry

Objective:To investigate the effect of combined detection of serum carcinoembryonic antigen(CEA),cytokeratin 19 fragment(CYFRA21-1),cancer antigen 125(CA125),and neuron-specific enolase(NSE)in patients with lung cancer by fluorescence flow cytometry.Methods:From August 2019 to July 2022,200 patients with lung cancer diagnosed by pathology in our hospital were retrospectively analyzed.2 mL venous blood was collected in a fasting state and centrifuged to separate the serum(containing human chorionic gonadotropin antibody[anti-hCG antibody],hepatitis B surface antibody[anti-HBs antibody],and CEA).Results:The sensitivities of CEA and CYFRA21-1 detected via enzyme-linked immunosorbent assay(ELISA)were 100%,and the detection limits were 0.5 ng/mL and 0.1 ng/mL,respectively;the sensitivities of CA125 and NSE detected via flow cytometry were 100%,and the detection limits were 10 U/mL and 2 ng/mL,respectively.Compared with ELISA,the sensitivities of CA125 and NSE detected via flow cytometry were higher.When the concentration of CEA was 10-40 ng/mL,the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 40-80 ng/mL,the sensitivity of CEA significantly decreased(P<0.01),but the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 80-200 ng/mL,the sensitivities of all four markers showed no significant changes(P>0.05).Conclusion:Compared with the double-antibody sandwich ELISA,fluorescence flow cytometry has certain advantages,including high sensitivity,good precision,short detection time,low sample usage,and low medical cost;thus,it is worthy of clinical promotion.

The Clinical Value of Detecting the Level of Exfoliated Cells in Pleural Effusion by Flow Cytometry in the Differential Diagnosis of Non-

Objective:To explore the value of flow cytometry(FCM)in detecting the level of exfoliated cells in pleural effusion in the differential diagnosis of non-small cell lung cancer and benign lung diseases.Methods:Clinical data of patients with non-small cell lung cancer who were hospitalized in Hebei hospital from June 2019 to March 2022 were collected.A total of 98 patients were included,and 63 patients with alveolar lung disease were screened during the same period,and the two groups of patients were analyzed.Results:Compared with alveolar lung disease group,FCM detection and analysis showed that the level of exfoliated cells in the pleural effusion of non-small cell lung cancer(NSCLC)patients was 99(3-969)/100,000,and patients with alveolar lung disease was 4(0~19)/100,000.Additionally,compared with the alveolar lung disease group,the level of exfoliated cells in the pleural effusion of patients with non-small cell lung cancer(NSCLC)was significantly increased(P<0.001).The diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in non-small cell lung cancer was assessed using ROC curves and using 95%CI(-11.1,-13.2)with a sensitivity of 0.75 and specificity of 0.94,and the diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in alveolar lung disease was assessed using 95%CI(-11.1,-13.2)with a sensitivity of 0.71 and specificity of 0.87.Conclusion:Flow cytometry has a wider range of clinical applications,simple operation,low cost,and high sensitivity,which makes it of great significance in disease diagnosis.

PCR-Flow定量检测外周血嵌合体方法的建立及临床应用

目的:建立能定量检测肾移植术后供体来源细胞(嵌合体)的PCR-Flow方法。 方法:利用细胞内聚合酶链反应(PCR)结合流式细胞仪、琼脂凝胶电泳、凝胶成像分析、荧光显微镜等技术,用两位人类白细胞DR抗原(HLA-DR)位点不同献血者外周血单核细胞,模拟供受体细胞混合(嵌合体),进行实验可行性和准确性研究,并对实验进行特异性分析。在此基础上对本院1999年1月至2001年12月进行肾移植联合供体骨髓细胞(donor bone marrow cell, DBMC)输注36例(DBMC组)及未进行骨髓输注的同一供体配对患者26例(配对对照组)移植术后嵌合体进行动态检测。 结果:模拟的嵌合体百分比与PCR-Flow检测嵌合体百分比呈直线关系,且方法的敏感性为0.05%,为特异性扩增。DBMC组术后4周和8周嵌合体与配对对照组相比明显增加,分别为(9.208.03)%与(1.292.05)% (P<0.01),和(7.73 7.35)%与(0.761.93)%(P<0.01)。但8周以后各个时间点两组之间无统计学差别。 结论:PCR-Flow检测嵌合体方法可靠、敏感性和特异性高,是检测外周血中微量嵌合体的新方法。

甘薯曲叶病毒PCR-层析试纸条快速检测方法的建立

为了能够快速且同时大批量检测甘薯样本是否感染甘薯曲叶病毒(SPLCV),本研究建立了SPLCV cp基因的PCR-层析试纸条快速检测方法。首先,本研究构建SPLCV cp基因的重组质粒作为阳性对照,并对标记的特异性引物的扩增条件进行优化。结果显示,退火温度为57℃时,特异性扩增效果最佳;特异性检测结果表明,该引物对与甘薯无症状1号病毒(SPSMV-1)、甘薯杆状DNA病毒B(SPBV-B)、甘薯羽状斑驳病毒(SPFMV)、甘薯褪绿矮化病毒(SPCSV)和甘薯潜隐病毒(SPLV)均无交叉反应,表明该引物对具有较高的特异性。其次,本研究构建了PCR-层析试纸条快速检测系统,当抗体包被磁粒时,20μg地高辛单克隆抗体为0.1 mg羧基磁粒的最佳抗体标记量。特异性扩增产物在新构建的层析系统中3 min左右进行可视化检测;灵敏度检测结果表明,该层析系统的最低检测限为0.0001 ng·μL^(-1)基因组DNA,灵敏度是普通凝胶检测的10倍,说明该系统灵敏度较高。综上,本研究建立的PCR-层析试纸条检测系统具有可视性、灵敏度高、简便快速的特点,且可以同时对大批量的样本进行SPLCV检验,满足了脱毒薯苗上市前检测量大的需求。

Radio-protective effect of vitamin E on spermatogenesis in mice exposed to γ-irradiation: a flow cytometric study

Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE, D-α-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of γ-irradiation. The animals were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-α-tocopheryl acetate levels were 47.4 ± 3.2 μg/dL in the treated group, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 post-irradiation. The primary spermatocytes : spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid : spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation. The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of γ-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells from radiation.

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Application of CD45/SSC Gating Multiparameter Flow Cytometry in the Classification of Acute Leukemia——An Analysis of 139 Cases

In order to study the significance of flow cytometry immunophenotyping in the diagnosis of acute leukemia, CD45/SSC gating multiparameter flow cytometry (FCM) was utilized to analyze the immunophenotypes of 139 cases of acute leukemia. 139 cases of acute leukemia were enrolled in our hospital from April 1998 to April 2000. Morphological analysis and FCM immunophenotypic tests were conducted on all cases. Our results showed that CD45/SSC gating multiparameter flow cytometry immunophenotyping could reflect the origin of leukemic cells specifically. It is one of the important methods for the diagnosis of ALL, AML, and HAL. CD45/SSC gating multiparameter FCM analysis is a good technique for immunophenotyping. FCM immunophenotypic analysis can help improve the diagnosis and classification of acute leukemia, and extend the use of FCM in clinical practice.

Flow cytometric analysis of DNA content for four commercially important crabs in China

The genome size（C-value） of an organism is referring to the DNA content of its non-replicated haploid chromosome complement,generally deduced from measuring somatic diploid nuclei.We presented genome size（C-value） data obtained by flow cytometry for four commercially important crabs（Portunus trituberculatus,Charybdis japonica,Scylla paramamosain,and Eriocheir sinensis） common in the coast of China.Gallus domesticus（2C=2.5 pg） was used as the internal standard.The results showed that the C-value for P.trituberculatus,C.japonica,S.paramamosain,and E.sinensis were（2.31±0.01） pg,（2.33±0.03） pg,（1.64±0.02） pg,and（2.29±0.03） pg,respectively.The C-value of P.trituberculatus,C.japonica and S.paramamosain were reported for the first time.The data represented by the four species indicated that they had lower DNA contents than average DNA values in crustaceans（（4.99±0.48） pg）,and three of the four values were very similar if not identical.The results provide useful data for future studies in the fields of biodiversity,species conservation,and phylogeny of these commercial crabs.They will also be helpful in instructing the hybridization breeding program and estimating the cost of the whole genome sequencing project.

Genome Size of Alexandrium catenella and Gracilariopsis lemaneiformis Estimated by Flow Cytometry

Flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

Demands and technical developments of clinical flow cytometry with emphasis in quantitative,spectral,and imaging capabilities

As the gold-standard method for single-cell analysis,flow cytometry enables high-throughput and multiple-parameter characterization of individual biological cells.This review highlights the demands for clinical flow cytometry in laboratory hematology(e.g.,diagnoses of minimal residual disease and various types of leukemia),summarizes state-of-the-art clinical flow cytometers(e.g.,FACSLyricTMby Becton Dickinson,DxFLEX by Beckman Coulter),then considers innovative technical improvements in flow cytometry(including quantitative,spectral,and imaging approaches)to address the limitations of clinical flow cytometry in hematology diagnosis.Finally,driven by these clinical demands,future developments in clinical flow cytometry are suggested.

Characterization of ploidy levels in Chrysanthemum L. by flow cytometry

Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthe- mum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu moun- tain （Henan province）, and C. zawadskii was collected at Huangshan mountain （Anhui province）. We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to bea simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.

Analysis of DNA Content of Various Types of Spermatogenic Cells in Rat after Testicular Heating with Flow Cytometry

To measure DNA contents of spermatogenic cells and analyze the efficiency of spermatogenesis after testicular heating in rat Methods Eighty adult male Sprague-Dawley rats were randomly divided into experimental group （43 ℃, 30 min） and control group （22 ℃, 30 min）. According to day 0.5, 1, 3, 6, 10, 25, 35 and 50 after local testicular heating, every group was divided into 8 subgroups： experimental subgroups （n=6） and control subgroups （n=4）. DNA contents of various types of germ cells were observed with flow cytometry （FCM） in all groups. Results Compared with control groups, percentages of 4C cell （primary spermatocyte） in 0.5-35 d groups and percentages of 1C cell （spermatid and sperm） in 6-50 d groups significantly decreased in experimental groups （P〈0.05）, and percentages of 2C cell （spermatogonium and second spermatocyte） in 3 -35 d experimental groups increased significantly after heating （P〈0.05）. 4C：2C in all of 8 experimental groups and 1C：2C in 3-35 d experimental groups were down （P〈0. 05）, and in 1 d experimental group 1C：4C was up after heating （P〈0.05）. Conclusions After being heated, the number of spermatocyte firstly decreased, and then that of spermatid and sperm decreased too. Heat influences several stages in spermatogenesis and results in suppression of spermatogenesis. Flow cytometry is an effective method for researching on the change of spermatogenesis and has significance on mechanism about changing of spermatogenic cells induced by heat.

Flow Cytometric Analysis of DNA Content in Parotid Tumor and Its Contiguous Acini

Summary: To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1. 369, S % 16. 95, PI 26. 18 in malignant tumors; DI was 1. 171, S % 12. 41, PI 15. 54 in recurrent pleomorphic adenoma; DI was 1. 141, S % 12. 74, PI 13. 07 in pleornorphic adenoma, DI was 0. 999, S % 5. 10, PI 8. 00 in normal acini. Analysis of variance showed there was a significant difference (P<0. 01 ). The average DNA contents of shallow on deep lobe of contiguous tumors was 1. 08 in DI, 10. 65 in S %, 13. 49 in PI in malignant tumor, 1. 06 in DI, 8. 96 in S % and 9. 85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P>0. 05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation, which might play an important role in recurrent or malignant change of the parotid tumors.

Recent advances in fuorescence-based in vivo flow cytometry

The fuorescence-based in vivo flow cytometry(IVFC)is an emerging tool to monitor eirculating cells in vivo.As a noninvasive and real-timne diagnostic technology,the fluorescence based IVFC allows long-term monitoring of circulating cells without changing their native biological environment.It has been applied for various biological applications(eg,monitoring circulating tumor cells).In this work,we will review our recent works on fluorescence-based IVFC.The operation principle and typical biological applications will be introduced.In addition,the recent advances in IVFC flow cytometry based on photoacoustic effects and other label free detection methods such as imaging based methods,difuse-light methods,hybrid multimodality methods and multispectral methods are also summarized.

Flow Cytometric Detection of Circulating Tumor Cells in Breast Cancer Patients: A Blinded Study

Circulating tumor cells are cells that detach from the primary tumor site and migrate to the bone marrow or other tissues where they can initiate a metastatic site. Liquid biopsies are an emerging tool in the past decades that enables us to detect Circulating Tumor Cells in patients’ blood. Flow cytometry is a powerful tool used in liquid biopsy diagnostics. This aims to prove the sensitivity and specificity of a flow cytometric panel for the detection of CTCs in breast cancer patients using healthy individuals’ samples as controls. The study was blinded to the data analyzing researcher. Statistical analysis followed and results show 86.9% area under the curve which indicates that the particular method can be very promising for diagnosing breast cancer.