Method of transfection of STOP gene lentiviral vector into oligodendrocyte precursor cells of BTBR mouse model of autism / 解剖学报

Objective To investigate the effect of microtubule binding protein STOP on myelin formation of oligodendrocyte in BTBR mice spectrum disorder in vitro, a highly purified primary culture method of oligodendrocyte precursor cells from cerebral cortex of BTBR mice was established. Establishment of a highly efficient transfection method for overexpression of STOP gene in oligodendrocyte precursor cells of BTBR mice cerebral cortex using lentiviral vector. Methods BTBR mice were used as experimental objects, 6-10 suckling mice were taken each time, repeat 3 times independently. The single cell suspension was prepared by trypsin digestion, and the primary oligodendrocyte precursor cells were obtained by immunomagnetic bead cell sorting method . After 5 days of culture, the cell purity was identified by oligodendrocyte precursor cell marker staining. The primary cultured oligodendrocyte precursor cells were transfected with STOP gene vector constructed in the early stage of the project group. 72-96 hours after transfection, the fluorescence staining of oligodendrocyte precursor cells was observed under fluorescence microscope, and the transfection rate and cell survival rate were calculated. Results The oligodendrocyte precursor cells of BTBR mice extracted by immunomagnetic beads sorting method basically adhered to the wall completely after 48 hours, and the cells had strong ability of proliferation. On the fifth da)' of culture, the purity of the cells was more than 95% identified by immunofluorescence. A lentivirus transfection method for primary oligodendrocyte precursor cells of BTBR mice with high transfection efficiency was established. The fluorescence expression of the cells was obvious after being photographed by high connotation microscope, the lentivirus transfection rate of oligodendrocyte precursor cells was increased to 60%-70%. Conclusion The primaiy oligodendrocyte precursor cells of BTBR mouse cerebral cortex with high purity were successfull)' isolated and cultured. A method for lentivirus infection of primaiy oligodendrocyte precursor cells in the cerebral cortex of BTBR mice is successfully established.

Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting / 中华创伤杂志(英文版)

PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

In vitro culture and purification of Schwann cells: A systematic review / 中国组织工程研究

OBJECTIVE: Schwann cells can promote the regeneration of damaged peripheral nerves and serve as seed cells in the engineering repair of peripheral nerve tissue. The effective culture and purification of Schwann cells are the basis of clinical treatment for peripheral nerve injury. This paper summarized the literature of culture of Schwann cells in vitro in recent ten years and made a descriptive review on the research progress of Schwann cell culture and purification, in order to provide references for culture of Schwann cells in vitro. METHODS: Literature related to Schwann cell isolation, culture and purification was retrieved from PubMed, Web of Science, Medline databases, CNKI, Wanfang, VIP and other databases. The keywords included “Schwann cells; isolation; culture; purification”. All the papers obtained from the search were read, analyzed and judged, and finally 62 papers meeting the standards were included. RESULTS: The classical methods of Schwann cell culture include tissue block culture and enzyme digestion. Purification methods include mitosis resistance, immune selection, specific adhesion, pre degeneration, cold jet, differential adherent, laminin package, low serum, stimulating factor, fluorescence activated cell sorting or magnetic activated cell sorting, immunopanning, and extracorporeal shock wave treatment. CONCLUSION: Schwann cells can be cultured and purified in various ways in vitro, and each method has its advantages and disadvantages. How to obtain high-purity Schwann cells quickly and efficiently is still a challenge. Multiple methods can be combined for purification and affective factors can be controlled for Schwann cell proliferation as much as possible.

Advances of high-throughput screening system in reengineering of biological entities / 生物工程学报

Enzymes and cell factories are the core of industrial biotechnology. They play important roles in various fields such as medicine, chemical industry, food, agriculture, and energy. Usually, natural enzymes and cells need to be engineered to improve the catalytic efficiency, stability and enantioselectivity. Directed evolution makes it possible to rapidly improve the properties of enzymes and cell factories. Sensitive and reliable high-throughput screening approaches are the key for successful and efficient engineering of enzymes and cell factories. In this review, we first summarize the advantages and disadvantages of different screening methods and signal generation strategies as well as their application scope; we then describe the latest advances of ultra-high throughput screening technology applied in the directed evolution of enzymes and cell factories in the past three years. On this basis, we discuss the limiting factors that need to be further improved for high-throughput screening systems and forecast the future development trends of high-throughput screening methods, hoping that researchers in various fields including biotechnology and instrument development can cooperate closely to enhance the reliability and applicability of the high-throughput screening techniques.

Epidural Fat-Derived Mesenchymal Stem Cell: First Report of Epidural Fat-Derived Mesenchymal Stem Cell

STUDY DESIGN: Experimental study. PURPOSE: To determine whether epidural fat (EF) tissue contains mesenchymal stem cells (MSC). OVERVIEW OF LITERATURE: Spine surgeons are unaware of the contents of EF tissue and the reason for its presence between the ligamentum flavum and the dura mater; therefore, EF tissues are routinely eliminated during surgical procedures. However, EF removal causes certain postoperative problems, such as post-laminectomy syndrome. We hypothesized that the EF tissue may play a significant supportive role for the neural structures and other nearby conditions. METHODS: EF tissues were obtained from consenting patients (n=3) during posterior decompression surgery of the lumbar spine. The primary cells were isolated and cultured as per previously described methods with some modifications, and the cell morphology and cumulation were examined. Thereafter, reverse transcription–polymerase chain reaction (RT-PCR), a fluorescence-activated cell sorting (FACS) analysis, and differentiation potency for differentiation into osteoblasts, chondroblasts, and adipocytes were investigated to identify whether the cells derived from EF are MSC. RESULTS: The cells from the EF tissue had a fibroblast or neuron-like morphology that persisted until the senescence at p18. MSC-specific genes, such as OCT4, SOX2, KLF4, MYC, and GAPDH were expressed in the RT-PCR study, while MSC-specific surface markers such as CD105, CD90, and CD73 were exhibited in the FACS analysis. The differentiation properties of EF-MSC for differentiation into the three types of cells (osteoblast, chondroblast, and adipocyte) were also confirmed. CONCLUSIONS: Based on the cell culture, FACS analysis, RT-PCR analysis, and differentiation potent outcomes, all the features of the cells corresponded to MSC. This is the first study to identify EF-MSC derived from the EF tissue.

Condition optimization of the flow cytometry MoFlo Astrios EQ on single-cell sorting in 96-well plate / 上海交通大学学报（医学版）

Objective • To optimize the method of the flow cytometry MoFlo Astrios EQ on single-cell sorting in 96-well plate. Methods • Using different aperture nozzles and sorting ways, the 32D, U937, iBMDM and 293T cells were used for single-cell sorting after the precise adjustment of the instrument and various parameters. The hole numbers with single cell and single-cell clones were detected after sorting. Results • In the single-cell sorting mode, the hole numbers with single cell were 83-91 by 70 μm nozzle and 87-93 by 100 μm nozzle. After 7-10 days of culture, the hole numbers with single-cell clones were 36-58 by 70 μm nozzle. In 100 μm nozzle, the hole numbers with single-cell clones were 53-78 by electrostatic charge sorting and 69-81 by straight-down sorting, respectively. Conclusion • In single-cell sorting, a better cell viability and higher cloning rate are observed in 100 μm nozzle and straight-down sorting.

Single B cell sorting-based amplification and functional identification of HIV-1 monoclonal antibody genes / 中华微生物学和免疫学杂志

Objective@#To amplify and identify monoclonal antibody genes from HIV-1-infected patients.@*Methods@#Single cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay.@*Results@#The heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml.@*Conclusion@#In this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification.

Immortalization of spleen fibroblastic reticular cells infected by severe fever thrombocytopenia syndrome virus / 中华实验和临床病毒学杂志

Objective@#To isolate, purify and culture fibroblastic reticular cells (FRCs) of mouse in spleen, to develop a reliable and robust method to immortalize primary mouse FRCs, to filter stable FRCs cell lines, to prove that the clones can be infected by SFTSV in vitro.@*Methods@#After purifying FRCs by fluorescence activated cell sorting (FACS) from autoMACS-enriched stroma cells of mouse spleen, we infected FRCs by simian virus 40 large T antigen in vitro, screened the FRCs clones with puromycin, compared primary and immortalized FRCs by RNA sequencing(RNA-seq) technology, infected the clones with severe fever with thrombocytopenia syndrome virus (SFTSV) in vitro.@*Results@#We succeed in culturing purified FRCs from spleen, isolated four stable FRCs clones, two of which have a purity of 99%, survived for more than 50 passages, express the key FRCs marker podoplanin and do not express CD31 and CD45. Clone 01 lost the typical FRCs-like morphology, the rate of expansion of which is quite different from that of primary FRCs and Clone 02. Clone 02 can be infected with SFTSV, which has the same gene expression pattern and immunophenotype with primary FRCs.@*Conclusions@#The stable FRCs clone Clone 02 has FRCs-like morphology and express key FRCs surface markers podoplanin (GP38 or PDPN) and do not express endothelial cell markers CD31 and leukocyte common antigen CD45. The RNA expression profiles identified by RNA-seq are also characteristic of FRCs. Infected with SFTSV in vitro, Clone 02 will be a new platform to study SFTSV.

Advances and challenges in therapeutic monoclonal antibodies drug development

The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic

Screening and identification of human monoclonal antibodies to hepatitis E virus capsid protein / 中国免疫学杂志

Objective:To establish a method for rapidly screening human antibodies recognizing HEV capsids proteins from pe-ripheral blood.The antibodies recognizing HEV capsids proteins were screened from the peripheral blood of vaccinator and the properties of the antibodies were analyzed.Methods:The HEV capsids proteins specific memory B cells in peripheral blood were obtained by flow cytometry sorting.Then antibody variable genes were acquired through single-cell RT-PCR and recombined to express in eukaryocyte.Finally,the properties analysis of recombinant expressed human monoclonal antibodies were carried out.Results:Six hu-manized monoclonal antibodies recognizing HEV capsids proteins were successfully obtained,and most of them had binding activity and neutralizing activity.Conclusion:The sequence of human monoclonal antibodies recognizing HEV capsid proteins is successfully screened and successfully expressed in the eukaryocyte.The properties of the antibodies are identified,which lay the foundation for studying antibody evolution in the human body after vaccination.

Isolation and identification of neutralizing monoclonal antibodies to two Tier 2 viruses in a patient with HIV-1 infection / 中华微生物学和免疫学杂志

Objective To isolate neutralizing monoclonal antibodies to Tier 2 viruses in a Chinese patient with HIV-1 infection. Methods Monoclonal antibodies were isolated using single B cell sorting and monoclonal antibody expression technique. The international ImMunoGeneTics database (IMGT) was used to analyze antibody variable region genes. Antibody binding ability and neutralizing activity were tested by enzyme linked immunosorbent assay and TZM-bl/pseudovirus assay,respectively. Results Two monoclonal antibodies (E11 and H2) that could neutralize two Tier 2 viruses were isolated from the patient with clade B HIV-1 infection. Heavy chains of E11 and H2 were derived from IGHV4-4*08 with a somatic mutation rate of 15.79% and 14.74%,respectively. Light chains were both derived from IGKV3-15*01 with a somatic mutation rate of 8.33% and 7.95%, respectively. E11 and H2 could bind to HIV-1 clade B, CRF01_AE and CRF07_BC viruses. The half maximal inhibitory concentration(IC50) values of E11 and H2 were 18.78 μg/ml and 22.43 μg/ml against 398-F1 virus and 43.35 μg/ml and 39.45 μg/ml against 25710 virus. Conclusion In this study, two neutralizing monoclonal antibodies to two Tier 2 viruses were identified in the patient with HIV-1 infection,which might provide reference for the development of AIDS vaccines.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells

The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1⁻ and SSEA-1⁺ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice / 生物工程学报

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting / 대한생식의학회지

OBJECTIVE: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. METHODS: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. RESULTS: The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). CONCLUSION: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

CPC derivedexosome protects cardiomyocytes from oxidative stress / 中国药理学通报

Aim To explore the anti-apoptotic function of cardiac progenitor cells(CPCs)-derived exosome in vitro.Method CPCs were isolated from mouse heart using Magnetic Cell Sorting(MACS)system.Flow Cy-tometry(FC)determine the purity of stem cell surface antigen-1 positive(Sca-1 +)CPCs.Exosome was puri-fied from conditional medium,and confirmed by West-ern blot using CD63 as a marker,Nanoparticle Traffic-king Analysis(NTA)was used to detect the diameters and concentration of exosome.Then the cells were di-vided into control groups and CPC-exosome pre-protec-tion groups.H2 O2 was added into H9c2 cells to induce oxidative stress.Western blot was adopted to determine the expression of cleaved caspase-3.Results ① Im-munofluorescence showed that CPCs isolated by MACS were positively expressing Sca-1 protein;FC analysis showed that typical purity of Sca-1 +CPCs from the first preparations was more than 95%.② WB demonstrated that CD63 of exosome isolated from CCMwas positively expressed,and NTA results showed that the diameters of exosome were (82.33 ±3.06)nm(n =3).Micro-scope detected PKH-26 labeled exosome appeared in the cytoplasma of H9c2 cells.③ Western blot showed the CPC-exosome pre-protection groups significantly down-regulated the levels of cleaved caspase-3 com-pared to the control groups(P <0.05).Conclusion CPC can secrete exosome which carries many important cargos,which can effectively gather in H9c2 cells. CPC-exosome can protect H9c2 cells from the oxidative stress induced by H2 O2 .Our results highlight a new perspective strategy for cardiac disease.

Application of CellaVision DM96 in nucleated cells classification of serous cavity effusion / 国际检验医学杂志

Objective To explore the capacity of the Sweden CellaVision DM96 automatic digital cell morphology analysis sys‐tem (DM96) in nucleated cell classification of serous cavity effusion .Methods 36 specimens of serous cavity effusion were selected from the inpatients of Second Affiliated Hospital of Xi′an Jiaotong University in March 2015 and performed the Wright staining by the two kinds of method ,the Japanese Sysmex SP‐1000Ⅰ automatic smearing machine and manual smearing ,after staining ,the smear was performed the nucleated cells classification by DM96 .The consistency and relevance of the classification results by DM 96 with those by the Sysmex XT‐4000i were calculated .Results The classification results by DM96 had better consistency with the results by XT‐4000i ,moreover the cell images taking by DM96 were clear with high automatic degree .Conclusion The DM96 auto‐mated digital nucleated cell morphology analysis system is reliable and effective ,and has a significance for improving the cellular morphological analysis of serous cavity effusion specimen .

CD137 molecule permits single cell sorting and expansion of HIV-specific T cells in vitro / 中华微生物学和免疫学杂志

Objective To isolate HIV-specific T cell clone and to expand them in vitro through the activation-induced expression of CD137 molecule.Methods The peripheral blood mononuclear cells were isolated from HIV-infected patients and HIV Gag specific CD3+CD8+CD137+T cell subset were sorted to 96-well plate in 1 cell/well by multicolor flowcytometry and single cell sorting.After 14 days in vitro culture with feeder cells and cytokines, the numbers and phenotypes of the cultured HIV-specific T cells were calcu-lated and identified.Results The CD137 expression was low on rested T cells but up regulated by the stim-ulation with Gag peptide pool.The CD8+CD137+T cells could secret IFN-γ.The number of CD8 T cells reached to 106 after 14 days in culture and expanded to 107-108 cells after 28 days of culture in vitro 100%of the cells remained activated upon Gag stimulation.Conclusion In stead of using IFN-γ, CD137 could be utilized as a novel molecule to isolate and expand HIV specific T cells in vitro.The expanded antigen spe-cific T cell clones could maintain good activation status.