基于微流控芯片多波长荧光检测体系

利用四个LED分别匹配相应的激发滤光片,带通发射滤光片和PMT,自行搭建微芯片多波长荧光检测系统。用于复杂生物混合样品:四类试样(荧光胺标记的牛磺酸(FT Ex/Em 390/446nm)、荧光素(Fl Ex/Em 480/520nm)、5(6)-羧基-X-罗丹明(ROX Ex/Em 563/600nm)和花菁染料(Cy 5 Ex/Em 635/677nm)的同时分离和测定并得到充分的验证。     

辣椒素的荧光分析方法研究

荧光分光光度法可用于辣椒及高纯度辣椒素样品中辣椒素的定量分析。在Ex为278 nm,Em为312 nm荧光条件下,辣椒素在0.58-5.8μg/mL浓度范围内其浓度C(μg/mL)与荧光强度I具有良好线性关系,回归方程c=1.0377×10-3I-0.3667,R=0.9994,精密度RSD=0.08%(n=5)。平均回收率95.39%。     

基于双荧光和单荧光探针的溶酶体pH值测定方法比较

目的:通过比较基于双荧光探针和单荧光探针的2种溶酶体pH值测定方法,探究一种不依赖四甲基若丹明(TMR)的、基于异硫氰酸荧光素(FITC)单荧光的检测溶酶体pH值方法的可行性。方法:利用人肺癌肺泡基底上皮来源的A549细胞,分别采用FITC/TMR双荧光探针法和FITC单荧光双激发法测定溶酶体pH值;通过计算相对荧光强度的比值FI_(FITC488)/FI_(TMR561)或FI_(FITC488)/FI_(FITC405),绘制pH值标准曲线,用于pH值测定。结果:相对于FITC在Ex488 nm/Em520 nm波长下的荧光强度对pH值极为敏感,其在Ex405 nm/Em422 nm波长下对pH值不敏感,可用于荧光矫正;不同pH值条件下,FI_(FITC488)/FI_(FITC405)比值基本呈线性关系。结论:FITC单荧光双激发法只须使用一种荧光探针即可实现pH值检测,较FITC/TMR双荧光探针法更不易产生系统误差,更适用于溶酶体pH值检测。     

用YO-PRO-1和PI联合染色定量检测细胞凋亡

经不同浓度staurosporine处理诱导凋亡的G7细胞样品,分别用YO-PRO-1/PI和AV/PI进行荧光染色,借助流式细胞仪检测凋亡情况,将两种检测方法得到的结果进行统计学分析显示,二者有显著的相关性(r=0.9659,P<0.01),且没有显著性差异(P<0.05);另外,上述凋亡细胞样品经YO-PRO-1/PI染色后在荧光显微镜下计数凋亡细胞比例的结果与AV/PI流式细胞仪的检测结果也有显著的相关性(r=0.9903,P<0.01),且没有显著性差异(P<0.05)。以上这些结果表明,用YO-PRO-1/PI对细胞进行染色、借助流式细胞仪和荧光显微镜均能准确地检测细胞凋亡,可替代AV/PI流式细胞仪方法用于细胞凋亡的检测。     

影响流式细胞术定量分析细胞DNA荧光强度的几个因素

本文对影响流或细胞术定量分析细胞DNA荧光强度的四种因素进行了研究,结果表明,(1)醛类固定剂对细胞DNA荧光有显著影响,乙醇是较好的固定剂。(2)样品之间细胞数相差3倍以上对荧光测定有影响。(3)细胞在乙醇中固定的时间长短对荧光影响不明显,但CV值增大。(4)EB为10—20μg/ml、PI为50μg/ml是最佳荧光染色浓度。     

薄层色谱-荧光分光光度法测定淫羊藿药材中淫羊藿苷的含量

目的 :建立了薄层色谱 荧光分光光度法测定淫羊藿药材中淫羊藿苷的含量。方法 :用 70 %乙醇水浴回流提取 ,用醋酸丁酯 甲酸 水 (1.3∶1∶1)在 10℃以下放置后的上层溶液为展开剂 ,展开 ,分离淫羊藿苷 ,在Ex=4 30nm ,Em=4 80nm条件下测定荧光强度。结果 :线性范围为 0 .8～ 4 .8μg ,相关系数r =0 .9973,平均回收率 97.4 8%。结论 :此法简单、灵敏、准确 ,重现性好。     

糯和非糯水稻籽粒发育过程中内源多胺的变化

本试验按Ma等的内标法(internal standard,InProceedings of Fourth Asian Chemical Congress.August,1991.Beijing)进行衍生提取测定,衍生化样品在高效液相色谱(HPLC)u-Bondapak NH_2(100μm3.9×300nm)柱进行分析,荧光检测(Ex=338nm,Em=495nm),峰面积积分定量。分析测定两对水稻(Oryzasativa)糯性等基因系原丰早和糯原丰早、广陆矮4号和糯广陆矮4号开花后7～30d期间,籽粒中腐胺(Put)、尸胺(Cad)、精胺(Spm)和亚精胺(Spd)4种多     

昆虫体内多胺的高效液相色谱(HPLC)测定

建立丹磺酰氯柱前衍生HPLC快速测定昆虫体内多胺含量的方法。以C18(250mm×4.6mm,5μm)为固定相,甲醇和水为流动相,梯度洗脱,柱温40℃,流速1mL/min,荧光检测波长激发波长(Ex)280nm,发射波长(Em)515nm,测得腐胺(put)、亚精胺(spd)和精胺(spm)三者回收率分别为98.7%,99.2%和97.8%,回归方程线性良好(r值均大于0.99),分析时间为16min。该法简洁、快速、灵敏度高、重现性好,可有效分析昆虫及其他生物样品中微量多胺的含量。     

羟基乙酰化姜黄素诱导的声动力治疗对巨噬细胞凋亡与坏死的影响

目的:探讨羟基乙酰化姜黄素(HAC)介导的声动力治疗对人急性单核细胞白血病细胞核(THP-1)源巨噬细胞凋亡的影响。方法:体外诱导THP-1源巨噬细胞,通过单纯药物和单纯超声对细胞存活率的影响优化出声动力治疗的最适条件。将细胞分为4组(1×105cells/ml):对照组、单纯超声组、单纯HAC组和声动力治疗组。应用声动力体外治疗巨噬细胞,药物HAC终浓度为5μg/ml,超声强度为0.5 W/cm2,超声时间为5 min。不同治疗方法后用无血清培养液培养6 h,用CCK-8法测定细胞存活率的变化,用Annexin V/PI法测定细胞凋亡、坏死变化。结果:CCK-8法测得声动力治疗后,细胞存活率明显下降(P0.01),Annexin V/PI法测得声动力治疗后,细胞凋亡率和坏死率均升高,凋亡/坏死比升高(P0.01)。结论:羟基乙酰化姜黄素介导的声动力治疗对THP-1巨噬细胞的凋亡有明显的诱导效应。     

SCP诱导人肝癌细胞凋亡与bcl-2基因表达的关系

目的探讨鲨鱼软骨制剂(SCP)诱导人肝癌细胞系(SMMC7721)凋亡的作用机制.方法以不同浓度SCP加入体外培养的SMMC7721细胞中,用MTT比色法检测细胞存活率;Hoechst33342/PI荧光染色,荧光显微镜分析凋亡细胞百分率;流式细胞术进行细胞凋亡定量;琼脂糖凝胶电泳检测DNA梯状条带;免疫细胞化学染色法检测Bcl-2蛋白的表达.结果 SCP明显抑制SMMC7721细胞生长,IC50值为1.25mg/ml;荧光显微镜下可见50%以上细胞为凋亡细胞的形态学改变;琼脂糖凝胶电泳呈现梯状条带(DNA ladder);免疫细胞化学检测显示SCP诱导人肝癌细胞凋亡过程中Bcl-2表达明显降低.结论 SCP诱导SMMC7721细胞凋亡,可能与下调Bcl-2表达有关.     

Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G₀/G₁, sub-S and sub-G₂/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.     

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.     

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death

BACKGROUND: Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. METHODS: Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. RESULTS: U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. CONCLUSIONS: These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Dual excitation multi- fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent "energy transfer" from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 micro g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.     

Comparative analysis of apoptosis in HL60 detected by annexin-V and fluorescein-diacetate.

BACKGROUND:Our aim was to compare and evaluate apoptosis formation as detected by propidium-iodide (PI)/annexin-V or PI/fluorescein-diacetate (FDA) as dose-response parameters in a human promyelocytic leukemia cell line, HL60. METHODS:In exponentially growing HL60 cells, apoptosis was induced by ionizing radiation, hyperthermia, topotecan, and cytosine beta-D-arabinofuranoside. At 4 consecutive days following induction, apoptosis was detected by double-labelling, either with PI/annexin-V or PI/FDA. Forward and side scatter, red (PI), and green (FDA or annexin-V) fluorescence were measured by flow cytometry. RESULTS:While light scatter discriminated between morphologically damaged and undamaged cells, fluorescence differentiated vital, apoptotic, and dead cells. Equal proportions of these three subpopulations were detected by both staining techniques. Occasionally, early and mature apoptoses were identified as distinct clusters. During the 4-day observation period, no pronounced maxima of the apoptotic fractions were obtained with either treatment modality. The gradual increases usually showed a delay of 1-2 days. CONCLUSIONS:FDA and annexin-V are equally suitable for detecting apoptosis. Separation improves with time after induction, indicating that, with respect to test specificity, mature apoptoses are superior to early stages. However, the sensitivity towards low rates of apoptosis after weak induction appears limited with both staining procedures.     

Towards an understanding of apoptosis detection by SYTO dyes.

BACKGROUND: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. METHODS: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. RESULTS: Loss of SYTO16 fluorescence (SYTOlow/PI+ events) has been observed in cells exposed to oncotic stimuli, whereas SYTOhigh/PI+ events did not prevail at any treatment scenario. We tracked similarities and discrepancies between SYTO16 and TMRM probes. Often, SYTO16 and TMRM exhibited the same staining profiles, as loss of their fluorescence was detected in a single cell population. However, both mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, appeared to induce distinct staining profiles of SYTO16 and TMRM, with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. Importantly, in both cases (FCCP and HA14-1) the decrease of SYTO16 fluorescence was blocked by pharmacological inhibition of caspases (with z-VAD-fmk). CONCLUSIONS: The data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of Deltapsim dissipation. Commonly observed similarities between SYTO and TMRM may stem from the fast kinetics of apoptotic events once cell death is initiated.     

Flow-cytometry assay for apoptosis using fluorophor 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange

Recently it has been shown that redox catalytic interactions of cytochrome c with cardiolipin (CL) and subsequent oxidation of CL occurs during apoptosis. Oxidation of CL is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. 10-N-Nonyl acridine orange (NAO), a fluorophor that forms a stable complex with reduced form of CL, but not with oxidized CL, can be used for flow cytometry analysis of this effect. It has been shown that after the incubation of CTLL-2 cells in the presence of 7% ethanol (90 min) and subsequent staining with NAO, a cell population with low intensity of fluorescence appears. Flow cytometry analysis of the cells by means of a conventional method (annexin V-FITC/propidium iodide (PI)) showed that the cell population with low intensity of NAO fluorescence corresponded to the population of apoptotic cells with good coincidence of percentages. Then apoptosis was induced in the cells of three lines by growth factor deprivation (IL-2-dependent cell line CTLL-2) or as a result of actinomycin D treatment, an RNA synthesis inhibitor (Jurkat and Raji cell lines). Comparison of the data obtained by a conventional assay (annexin V-FITC/PI) and a newly elaborated assay (NAO/PI) has demonstrated a good coincidence. The data obtained with these methods exhibited significant level of correlation (0.953, p < 0.0001).     

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability^1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells³. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells ^4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane ^1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases^7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence ^1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment¹⁰. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence¹⁰. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).     

Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr) in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at < 4 μM) and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60 ns, 13.2 kV/cm, 10 Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3 min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.