流式细胞术检测体内细胞毒效应方法的研究

目的 建立一种用流式细胞仪检测体内细胞毒效应的方法.方法 用不同浓度的羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记靶细胞和非靶细胞,以1:1的比例混合靶细胞和非靶细胞,尾静脉回输小鼠,16h后取小鼠脾脏淋巴细胞,流式细胞仪检测CFSE标记靶细胞和非靶细胞的比例,测定体内被杀伤靶细胞的百分率.结果 荧光染料CFSE能有效标记靶细胞,16h后流式细胞仪能很好地检测到体内被杀伤靶细胞的百分率.结论 CFSE荧光染料标记靶细胞并结合流式细胞仪分析的方法能有效地检测到体内细胞毒效应,该技术有望在肿瘤免疫和移植排斥的体内监测中得到广泛应用.     

流式细胞术和微量细胞毒法检测HLA-B27的比较

人白细胞相关抗原 (ＨＬＡ Ｂ2 7)分子 (简称Ｂ2 7)的生理功能是携带提呈的细菌多肽 ,并激活ＣＤ8+ Ｔ细胞。Ｂ2 7因与强直性脊柱炎 (ＡＳ)等脊柱关节病 (ＳＰＡ)的关联 ,是所有疾病与ＨＬＡ相关中最为密切的 ,多年来一直是风湿病学界研究的热点之一[1] 。分析Ｂ2 7的表达对ＡＳ的发病机制、诊断、预防及预后判断都有重要意义[2 ,3 ] 。既往采用微量细胞毒法检测ＨＬＡ Ｂ2 7表型 ,本文采用流式细胞术进行检测 ,并比较了两种方法的优缺点。1　材料和方法1.1　材料　微量细胞毒法用倒置显微镜产自重庆光学仪器厂。标准羊抗人血清、Ｂ2 7抗原…     

流式细胞术测定淋巴细胞的分裂

为确立流式细胞仪检测淋巴细胞的分裂的新方法 ,利用CFSE标记新鲜分离外周及胸腺淋巴细胞后 ,加入PMA、ConA、抗TCR抗体等培养 3d ,染色进行FACS分析。结果 ,诱导T细胞活化分裂的 3种刺激剂 ,以PMA刺激作用最强 ,ConA次之 ,抗TCR抗体最弱。表明CFSE标记细胞 ,流式细胞仪分析法不仅可以检测单细胞水平上细胞的分裂 ,还可根据荧光强度判断细胞的分裂次数。     

流式细胞术检测淋巴细胞增殖方法的建立

目的 :建立一种用流式细胞仪同时测定T淋巴细胞增殖与细胞因子分泌的方法。方法 :用非特异性刺激剂PMA、PHA刺激 1 0例正常人外周血单个核细胞 ,体外培养 2、4、6、8、1 0h ,流式细胞术在单细胞水平检测T淋巴细胞的功能性激活、表面活化标志性抗原 (CD69)的表达、DNA合成 (BrdU掺入法 )及胞内细胞因子 (IL 4、IFN γ)分泌。选择合适的刺激剂、调整细胞浓度、优化实验方法 ,确定最佳实验条件。结果 :BrdU掺入法测定T淋巴细胞增殖 ,最佳的反应细胞浓度为 1× 1 0 6ml-1 ,比较不同刺激剂和体外培养时间 :PMA(2 0ng ml)刺激后 8hT细胞中CD69+、BrdU+双阳性细胞比例最高 (82 3 %± 7 2 % ) ,IFN γ+、IL 4+细胞百分率分别为 2 5 2 %± 3 7%和 3 4%± 1 6 % ,均显著高于未刺激组 (P <0 0 1 )。结论 :应用流式细胞术进行单个核细胞的增殖分析可以同时检测细胞表面活化抗原的表达、DNA合成及胞内细胞因子的分泌。此方法能够分析不同亚群细胞对于不同刺激原的反应和激活表型。敏感性高、重复性好 ,可在单细胞水平检测单个核细胞的增殖和功能性激活     

基于流式细胞术评价单核细胞介导的抗体依赖性细胞介导的细胞毒效应的方法学建立和应用

目的：建立基于流式细胞术的评价单核细胞介导的抗体依赖性细胞介导的细胞毒效应的检测方法。方法 PKH26和CFSE染色的P815细胞为靶细胞,与P815特异性抗体孵育形成抗原抗体复合物,加入外周血单个核细胞作为效应细胞,共同孵育后流式细胞术检测 CD3－CD14＋PKH26＋CFSE－细胞群的百分比,并确定最佳效靶比及效应细胞和靶细胞的孵育时间。运用上述方法对23例HCV慢性感染者和22例健康人的单核细胞介导的抗体依赖性细胞毒作用（ antibody depend-ent cellular cytotoxicity , ADCC）进行比较分析。结果可通过流式细胞技术检测CD3－CD14＋PKH26＋CFSE－细胞群来评价单核细胞介导的ADCC效应,最佳效靶比为10∶1,最佳杀伤孵育时间为4 h。慢性HCV感染者单核细胞介导的ADCC效应较健康对照明显降低（ P＝0．009）。结论本研究建立了基于流式细胞术的单核细胞介导的ADCC效应的检测方法,为病毒感染及药物研发中免疫学评价提供快速、敏感、安全的检测手段。     

基于流式细胞术的scFv抗体库筛选技术的建立

目的 利用NlpA前导肽诱导抗体锚定细菌内膜建立筛选scFv抗体库的展示技术,为高亲和力抗体的筛选奠定基础.方法 从pNAD质粒中克隆出NlpA前导肽基因序列,酶切后将该序列克隆进pHEN1表达载体中.以PEAI质粒为模板,利用PCR的方法克隆得到抗-hIL-1β抗体的重链可变区和轻链可变区基因,然后利用重叠PCR的方法构建得到抗-hIL-1β scFv抗体.将scFv抗体插入到NlpA leader-pHEN1表达载体中构建成展示scFv的重组质粒pBSD-scFv.将pBSD-scFv转入到E.coli DH5α中诱导表达,原生质球制备后,采用梯度浓度的抗原进行孵育,最后经流式细胞术(FCM)检测抗体展示情况并且分选阳性群体,利用质粒提取的方法来替代PCR方法拯救阳性基因,转化E.coli DH5α,利用FCM再次检测该群体展示的抗体与抗原结合情况.结果 所展示的抗-hIL-1β scFv抗体依次与抗原和FITC标记的抗原特异性抗体孵育后,用FCM实时检测,结果显示出很强的荧光信号并且表现出抗原浓度依赖性.拯救出的pBSD-scFv-原生质球的FCM检测结果与首次展示的FCM结果一致,该系统能够稳定的展示抗体.结论 经过该细菌展示系统展示的scFv抗体能够有效的折叠,与相应的抗原具有很好的特异结合能力.     

三色法流式细胞术检测胞内细胞因子的建立

运用荧光标记的抗细胞表面抗原单抗及抗细胞因子单抗, 结合固定、破膜处理技术, 建立了三荧光染色法流式细胞术检测细胞内细胞因子的方法, 探讨了不同刺激剂及细胞培养时间的选择, 并对５ 例正常人产生ＩＦＮ γ、ＩＬ ４ 的ＣＤ４ 及ＣＤ８ 细胞进行测定。结果表明： 产生ＩＦＮ γ的ＣＤ４ 及ＣＤ８ 细胞明显多于产生ＩＬ ４ 的细胞, 并且产生ＩＦＮ γ的ＣＤ８ 细胞百分数比较ＣＤ４ 细胞相对增多; 同时产生ＩＦＮ γ／ＩＬ ４ 的ＣＤ４ 、ＣＤ８ 细胞较少。此方法可从单个细胞水平直接用于Ｔ细胞亚群及其细胞因子的检测。     

流式细胞术定量检测细胞凋亡

本文报道了几种药物处理人体外周血细胞后，用双染色法在流式细胞仪检测凋亡细胞的发生。将凋亡细胞分选出用琼脂糖核酸电泳ＤＮＡ分析，证实了流式细胞仪所测定的结果。比较了单染色法检测凋亡细胞的局限性．     

流式细胞术检测全血白细胞吞噬白念珠菌方法的建立和应用

目的建立全血白细胞吞噬白念珠菌的流式细胞术检测方法。方法绘制白念珠菌生长曲线后,取生长对数中期的白念珠菌,以活体细胞示踪荧光探针二乙酰羧基荧光黄琥珀酰亚胺酯(CFDA-SE)标记,与以PC5标记的CD45单克隆抗体预标记的全血白细胞,在37℃分别反应10、30、60min(MOI≈10∶1),以流式细胞术观察CD45阳性细胞对白念珠菌的吞噬情况。结果白念珠菌在液体酵母浸出粉胨葡萄糖培养基(YPD)中37℃、50mL/LCO2培养,第5~11h为对数生长期。10mmol/LCFDA-SE染色30min,可使白念珠菌均匀着色;CD45阳性细胞和标记的白念珠菌作用10、30、60min,CD45阳性细胞中中性粒细胞的吞噬率分别为(80.1±6.1)%、(83.8±7.7)%和(92.3±11.2)%;单核细胞的吞噬率为(11.2±3.6)%、(15.8±4.4)%和(27.7±6.8)%,淋巴细胞作为无明显吞噬作用内参,其吞噬率为(0.9±0.3)%、(0.8±0.4)%和(5.2±1.6)%。结论成功建立流式细胞术检测全血白细胞吞噬白念珠菌的方法,全血白细胞与白念珠菌共孵育30min是较为合适的作用时间。     

流式细胞术检测细胞内细胞因子的研究进展

细胞因子作为细胞间信号传导的分子, 主要介导和调节免疫应答及炎症反应, 刺激造血细胞生成和组织损伤的修复等。正常情况下, 细胞因子的表达和分泌受到机体严格地调控。在病理状态下, 细胞因子会出现异常表达。因此, 检测细胞因子对于某些疾病的诊断、治疗具有重要价值。当前,仅对细胞进行定量及活性检测已不能满足需要, 在单细胞水平研究细胞因子的表达更加重要。随着流式细胞术 (flowcytometry, FCM)在临床、科研中的广泛应用, 利用FCM与细胞内细胞因子 (intracellularcytokine, ICK)染色技术相结合,可提供一种有效地在单细胞水…     

A flow cytometry-based assay for quantitative analysis of cellular proliferation and cytotoxicity in vitro

A novel method based on flow cytometry (FCM), which can count the number of detected cells, has been developed for the evaluation of cellular proliferation and cytotoxicity in vitro. It provides a tool that directly counts cell number without being influenced by the metabolic state of the cells, discriminates target cells from effector cells in cell-mediated cytotoxicity assay, and with less treatment step and free radioactivity. In this paper, we have prepared the PG cells (a highly metastatic human lung cancer cell line) and peripheral blood lymphocytes (PBL) with various concentrations and ratios of concentration to validate the method. The results were compared with MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay and the regression analysis results showed that this method worked very well. We have also used this method to evaluate mitogen-induced proliferation and cytotoxicity. The results indicated that this method might yield high sensitivity and reliability.     

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. Theseassays are based on the principle that intact plasma membranes in live cells excludespecific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB)exclusion assay has limitations. The dye can be incorporated by live cells after ashort exposure time, and personal reliability, related to the expertise of theanalyst, can affect the results. We propose an alternative assay for evaluating cellviability that combines the TB exclusion test and the high sensitivity of the flowcytometry technique. Previous studies have demonstrated the ability of TB to emitfluorescence when complexed with proteins. According to our results, TB/bovine serumalbumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which isdetectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v)was defined as the optimum concentration for distinguishing unstained living cellsfrom fluorescent dead cells, and fluorescence emission was stable for 30 min aftercell treatment. Although previous studies have shown that TB promotes greenfluorescence quenching, TB at 0.002% did not interfere with green fluorescence inhuman live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonalantibody. We observed a high correlation between the percentage of propidiumiodide+CD3/FITC⁺ and TB+CD3/FITC⁺ cells, as well as similardouble-stained cell profiles in flow cytometry dot-plot graphs. Taken together, theresults indicate that a TB exclusion assay by flow cytometry can be employed as analternative tool for quick and reliable cell viability analysis.     

The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo

Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.     

Fluorescence polarization assay by flow cytometry

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.     

流式细胞术定量检测细胞凋亡3种方法的比较研究

目的：探讨流式细胞术定量检测细胞凋亡３种方法的价值。方法：同时使用ＰＩ染色，ＴＵＮＥＬ及Ａｎｎｅｘｉｎ Ｖ／ＰＩ３壹量检测地塞米松处理小鼠的胸腺细胞凋亡发生率。结果：ＰＩ染色，Ａｎｎｅｘｉｎ Ｖ／ＰＩ，ＴＵＮＥＬ３种方法凋亡检出率分别为２７．１９％，３２．２８％，５０．１７％，两者之间均有显著差异。     

流式细胞仪分离人早孕绒毛外滋养细胞及鉴定

目的用流式细胞仪分离早期绒毛外滋养细胞，以便对其功能进行进一步研究。方法用胰蛋白酶消化法得到早期绒毛细胞，用流式细胞仪分离纯化绒毛外滋养细胞。所得的细胞用免疫细胞化学，光镜，透射电镜检以及Westen-blot方法鉴定。结果使用流式细胞仪成功的分离了绒毛外滋养细胞，纯度超过98％。结论此方法可快速准确大量获得绒毛外滋养细胞。     

Evaluation of Rh monoclonal antibodies for flow cytometry tests

As participants of the “III International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens”, we tested 43 RH monoclonal antibodies by the flow cytometry technique. Besides the anti-D antibodies (not included in this paper), we tested the following antibodies: three (3) anti-C; one (1) anti-C^w; six (6) anti-c; eight (8) anti-E; three (3) anti-e; two (2) anti-G; one (1) anti-CcEe (total = 24 antibodies). These antibodies (from different lab sources) were tested against antigen positive cells (homozygous or heterozygous) and antigen negative cells. When available, some of them were tested against “rare” phenotypes like r_yr, r″^Gr, r^Gr, R₂R_z. All the three anti-C tested, showed poor discrimination between positive and negative cells; from the six anti-c tested, only three had good results (Workshop n° 18, 20, 21) with a superior performance of one of them (Workshop n° 18). From the eight anti-E tested, we found two (Workshop n° 139 and 153) with good performance; all the three anti-e were non reactive; the two anti-G failed to react with r′r red cells; the anti-C^w reacted better with R₁^wr cells than R₁^wR₁; and the anti-CcEe antibody showed good results with all the phenotypes tested. From the 24 antibodies tested, we found six (25%) antibodies with a good performance.