A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.     

A luminescent assay for natural cytotoxicity using K562 cells transfected with the plasmid producing alkaline phosphatase

A novel non-radioactive assay for natural cytotoxicity is described. A new target cell line was established by the transfection of K562 cells with a plasmid encoding DNA for secretion type of placental alkaline phosphatase(ALP). The assay was based on the kinetic determination of heat-stable ALP in the co-culture of the target cells with effector sample from human peripheral blood. The inactivated effector sample for the control prepared by irradiation(50Gy X-ray) or heat treatment(56 degrees C, 20 min), however, showed an enhancement effect on the ALP production in the target cells. This effect was depending on the concentration of monocyte component(adherent cells on plastic dish) by unknown reason, and was not reduced by the treatment for NK inactivation. Cytotoxic activity was therefore determined by the percent decrease in the ALP productivity compared to that in the control. The correlation coefficient between cytotoxicity values by this ALP synthesis assay and those by 51Cr release assay was 0.93. This cytotoxicity test is simple and unique in terms of estimation for protein synthesis affected in target tumor cells, although the mechanism of the monocyte(or macrophage) effect remained to be elucidated.     

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity.

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.     

三种比色法评价4种牙科金属材料对L-929细胞的毒性

背景：采用不同方法评价材料的细胞毒性可能会得出不同的实验结果。 目的：采用3种比色法评价镍铬合金、钴铬合金、3铬13及纯钛等牙科金属材料对小鼠成纤维细胞(L-929细胞)的细胞毒性。 方法：以镍铬合金、钴铬合金、3铬13及纯钛4种牙科金属材料的浸提液分别作用于体外培养的L-929细胞24,72 h。以体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阴性对照组,以0.7%丙烯酰胺+体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阳性对照组,分别采用MTT、CCK-8及结晶紫3种比色法检测上述材料的细胞毒性。 结果与结论：①4种材料浸提液中培养的细胞形态正常,胞内结构清晰,随着培养时间延长细胞大量增殖,与阴性对照组细胞形态无明显差异。阳性对照组细胞数量明显减少,形态完整性受破坏,形成大量细胞碎片。②培养24 h时,CCK-8比色法检测中钴铬合金组的细胞相对增殖率低于阴性对照组(P < 0.05),MTT及结晶紫比色法检测中钴铬合金组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05);培养72 h时,MTT比色法检测中4种牙科金属材料组细胞相对增殖率低于阴性对照组(P < 0.01),CCK-8及结晶紫比色法检测中4组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05),但材料细胞毒性均为0-1级。表明上述4种牙科金属材料细胞毒性均在临床应用的允许范围内,具有良好的生物安全性。     

Acquired spontaneous cytotoxicity of K562 in a subpopulation of monocytes

Human peripheral blood monocytes were placed on a discontinuous density gradient of bovine serum albumin and fractionated into five subpopulations. Cells from each subpopulation were assayed for spontaneous cytotoxic activity against K562 tumor cells. Immediately following fractionation, monocytes were not cytotoxic. Following incubation for at least 48 hr, monocytes from two layers of the gradients clearly exhibited greater spontaneous cytotoxic activity than all others. The degree of cytotoxicity expressed by cells of these layers was enhanced by the addition of indomethacin and inhibited by prostaglandin E2 (PGE2). Monocytes acquiring spontaneous cytotoxicity did not secrete measurable levels of PGE2 and had increased levels of purine nucleoside phosphorylase after 72 hs of culture in vitro. Surface markers HNK-1 and Mac-1 normally associated with cytotoxic function, were detected on these cells by indirect immunofluorescence at isolation and after culture. The fraction with the greatest cytotoxic activity showed an increase in the proportion of cells displaying reactivity to HNK-1 after culture compared to initial isolation.     

三种含硒化合物对K562细胞的抑制作用

目的 研究比较3种新的含硒化合物在体内、外的抗癌作用和作用机制. 方法 通过四甲基偶氮唑盐(MTT)比色法和移植瘤生长抑制实验及形态学观察、流式细胞术以及激光扫描共焦显微镜,检测分析含硒化合物对K562细胞的抑制作用. 结果 3种含硒化合物能明显抑制K562细胞增殖(P<0.05)和S180、H22移植瘤(n=10)的生长(P<0.01);使K562细胞出现体积缩小,细胞膜完整,染色质高度凝集,边集,核浓染、碎裂,伴有出泡现象和凋亡小体出现等典型的凋亡特征;对K562细胞的周期分布有明显影响,且在大剂量时出现了明显的亚二倍体峰;能够明显增加K562细胞内Ca2+、Mg2+和细胞内活性氧(ROS)的荧光强度(P<0.01),但pH值和线粒体膜电位显著降低(P<0.01). 结论 3种含硒化合物均有体内、体外抗肿瘤作用,其作用机制可能与Ca2+、Mg2+、(活性氧ROS)、pH值和线粒体膜电位(MMP)诱导的细胞凋亡有关.     

Visualization of the receptor for transferrin on K562 cells by a rosette-forming assay

A method is described to visualize the receptor for transferrin by a rosette-forming assay. K562 cells, a human pre-erythroid leukemia cell line, were used as a source of receptor-positive cells. Bovine erythrocytes coated with human transferrin by the CrCl₃ method were used as indicator cells. Rosette formation occurred at 37°C but not at 4°C, and was inhibited by soluble transferrin in a dose-dependent manner. Human lactoferrin, which is known to have considerable amino acid sequence homology with transferrin, did not inhibit rosette formation with transferrin-coated bovine erythrocytes. A variety of blocking and non-blocking monoclonal antibodies against the human transferrin receptor (42/6, B3/25 and OKT9) inhibited rosette formation. Rosettes are able to withstand low-speed centrifugation (130 × g, 2 min) making this method potentially useful for cytochemical identification of receptor-positive or receptor-negative cells. Also, it was possible to enrich receptor-positive cells by separation of rosette-forming from non-rosette-forming cells using a density gradient centrifugation technique.     

A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells

A solid-phase enzyme immunoassay for the detection of antibodies, specific for hemoglobin (Hb) is described. The application of glutaraldehyde resulted in a sensitive assay and allowed the use of urea, which is an important advantage if polypeptides not soluble in aqueous buffers are to be used. Mutation-carrying Hb chains can be purified, solubilized in urea and used in the immunoassay to monitor the purification and selection of antibodies specific for these variants. Specific antibodies are the main tools for the development of a hemoglobin-locus mutation system for detection of potentially mutagenic environmental agents. With erythrocytes as target cells, this system permits in vivo monitoring of subjects under exposure. Conventional antibody production, however, frequently turns out to be unsuccessful. The production of monoclonal antibodies has several advantages over conventional antibody production, but a sensitive antibody screening system is essential. Because of the sensitivity and the ease with which a large panel of antibody fractions against a vast panel of Hb antigens can be examined, the described immunoassay has potential value for the screening of hybridoma cultures.     

应用基因芯片研究As2O3诱导K562细胞前后差异表达基因

目的研究三氧化二砷(As2O3)处理K562细胞前后,基因表达的变化。方法提取As2O3作用K562细胞前后的总RNA,逆转录成cDNA并用Cy3标记对照组,Cy5标记处理组,与自制的包含162个cDNA片段的K562芯片杂交。结果K562细胞经As2O3作用后,162个基因片段的表达都有所降低,其中25个基因片段的表达降低较为显著(Cy5/Cy3<0.5)。这些表达下调的基因片段包括转导因子、代谢酶、信号通路蛋白、激酶等,有6个差异表达基因与细胞的凋亡通路密切相关。As2O3可能通过抑制胰岛素样生长因子的功能,诱导细胞凋亡。结论应用自制的基因芯片筛选到了As2O3诱导K562细胞前后差异表达的基因,主要与细胞凋亡密切相关。     

Evidence in support of the plasma membrane as the target for transferrin-adriamycin conjugates in K562 cells

We have used transferrin-adriamycin conjugates to deliver drug to transferrin receptors and have shown that the conjugates bind to and kill tumor cells in vivo and in vitro. This has been studied with the use of qualitative and quantitative assays. In this report, we present evidence indicating that the primary target of transferrin-adriamycin cytotoxicity is the plasma membrane. This was done using a spectrofluorometric method that takes advantage of the fluorescence properties of adriamycin. It was shown that, while DNA intercalation may be the primary mechanism of cytotoxicity for free adriamycin, transferrin-adriamycin conjugates were not observed to interact with nuclear DNA. This may be a useful consideration in the design of future chemotherapeutic studies with transferrin conjugates of anticancer drugs.     

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

Background  

This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the ⁵¹Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells.     

Hsp701A的RNA干涉诱导K562细胞凋亡

目的：研究RNA干涉（RNAi）Hsp701A基因的表达及其诱导慢性粒细胞白血病（慢粒）细胞株K562的凋亡。方法：构建Hsp701A基因小干涉RNA（siRNA）的真核表达载体，转染K562细胞并证实RNAi的有效性。用MTF比色法、AnnexinV-FITC／PI染色和流式细胞术，分别检测K562细胞的增殖、凋亡和细胞周期。结果：构建了Hsp701A siRNA的真核表达载体。将其稳定转染K562细胞后，RNAi组细胞中Hsp701 ARNA和蛋白的表达水平明显下降。Hsp701A siRNA能抑制K562细胞增殖并诱导其细胞凋亡，将其阻滞于G1期。结论：RNAi Hsp701A基因诱导的表达有望成为一种新的慢粒的治疗策略。     

Daxx抑制K562细胞向巨核细胞分化

目的:观察过表达死亡结构域相关蛋白(Daxx)对K562细胞活力和向巨核细胞分化的影响。方法:建立稳定过表达Daxx的K562细胞,荧光显微镜观察、实时荧光定量PCR和Western blot检测Daxx的过表达效果,CCK-8法检测过表达后细胞活力的变化;佛波酯(PMA)诱导K562细胞向巨核细胞系分化,Western blot检测在K562细胞向巨核细胞分化过程中Daxx和p-ERK的表达变化,流式细胞术检测CD41和CD61的表达变化;PMA处理过表达Daxx的K562细胞,NBT还原实验检测细胞分化情况,流式细胞术检测过表达Daxx后CD41和CD61的表达变化,Western blot检测p-ERK的蛋白水平。结果:建立了稳定的过表达Daxx的K562细胞,过表达Daxx抑制K562细胞的活力。PMA诱导K562细胞向巨核细胞分化,CD41和CD61表达水平增高,同时p-ERK的蛋白水平升高,Daxx表达水平下降。过表达Daxx可以抑制K562细胞向巨核细胞分化,CD41和CD61表达降低,同时p-ERK的蛋白水平降低。结论:过表达Daxx可以抑制K562细胞生长及向巨核细胞分化,同时抑制ERK的磷酸化。     

Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms-(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp -/- (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp -/- mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.     

地西他滨对K562/A02细胞阿霉素耐药性的影响

 目的: 观察地西他滨(DAC)对人慢性粒细胞白血病耐药细胞株K562/A02阿霉素(ADR)耐药性的影响,探讨其作用的可能机制。方法: 分别或联合应用不同浓度ADR和DAC作用于K562/A02细胞和其亲本细胞株K562,采用CCK-8法检测药物细胞毒性,Sequenom MassARRAY系统结合比色法评价DNA甲基化程度,流式细胞术检测K562/A02细胞细胞周期分布和细胞凋亡率。结果: K562/A02细胞较K562细胞具有显著ADR耐药性,前者ADR作用24 h的IC₅₀约为后者的50倍。而对DAC,在0.5~8 μmol/L作用浓度范围内,K562/A02细胞则较K562细胞更敏感。在相同ADR作用浓度(4.31和17.24 μmol/L)下,联合1 μmol/L DAC处理24 h能显著提高K562/A02细胞对ADR的敏感性,细胞存活率下降(P<0.05)。DAC和ADR均能影响K562/A02细胞的细胞周期进程和细胞凋亡率。1 μmol/L DAC的影响与作用时间相关,在作用24 h时以S期阻滞与细胞早期凋亡率升高为主,48 h时以G₂/M期阻滞与细胞晚期凋亡和坏死率升高为主。ADR则主要表现为浓度依赖性G₂/M期阻滞并诱导细胞晚期凋亡和坏死。两者联用使ADR对细胞周期分布的作用进一步加强,即表现为G₂/M期阻滞更加明显,但对细胞凋亡率的影响并无显著差异。而在基因组甲基化程度上,2种细胞没有显著差异,DAC作用前后也没有显著改变。结论: DAC能增强K562/A02细胞对ADR的敏感性,具有逆转耐药作用,其机制可能与调节K562/A02细胞细胞周期进程、促进细胞凋亡和坏死有关。     

Enhancement of lymphocyte-mediated K562 cytotoxicity by antibodies against complement membrane cofactor protein (CD46) and decay-accelerating factor (CD55).

A natural killer (NK) target, K562 (a human erythroblastoid cell line), was found by flow cytometry to express three complement regulatory membrane proteins on its surface: decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and p18 (CD59). We examined the effects of F(ab')2 of polyclonal antibodies raised against DAF and MCP and monoclonal antibody to p18 on the susceptibility of K562 to cytolysis by homologous lymphocytes, granulocytes and complement. C3bi-deposition was induced on K562 when the cells were treated with both anti-DAF and anti-MCP. Lymphocyte-mediated K562 cytolysis was markedly enhanced by these two antibodies whereas anti-p18 barely affected the degree of cytolysis. Complement immunocytolysis, on the other hand, became highest by combination treatment with the three antibodies, although anti-p18 and either anti-DAF or anti-MCP induced little potentiation of cytolysis. Granulocytes showed the least cytolysis and minimal potentiation of lysis by treatment with both anti-DAF and anti-MCP.     

蜂胶抑制白血病K562细胞增殖机制的初步研究

目的: 探讨蜂胶对K562细胞增殖、凋亡的影响及其作用机制。方法: 体外培养K562细胞,将不同浓度的蜂胶掺入细胞,采用MTT法检测细胞增殖抑制率,流式细胞术(FCM)检测细胞凋亡率,RT-PCR检测细胞中Nup98 mRNA表达水平。Western blotting检测细胞中Nup98蛋白表达水平。结果: 2 mg/L、20 mg/L和200 mg/L组蜂胶K562细胞的增殖抑制率和凋亡率明显高于对照组,并具有一定的时间和剂量依赖性。高浓度蜂胶作用后,Nup98 mRNA及蛋白表达均明显低于对照组。结论: 蜂胶可抑制K562细胞的增殖,诱导其凋亡,其机制可能与下调Nup98的表达有关。     

不同细胞因子诱导脐血来源的CIK、NK细胞对K562细胞杀伤活性的研究

目的：了解不同细胞因子组合扩增的脐血CIK、NK细胞对人K562细胞株的杀伤活性的差异性。方法：通过SCF、FLT3L、IL-2、IL-7及IL-15等细胞因子的不同组合扩增的脐血CIK、NK细胞，分为3组，A组（SCF＋IL-2＋IL-7＋IL-15）、B组（SCF＋FLT3L＋IL-2＋IL-7＋IL-15）和C组（IL-2＋IL-7＋IL-15，对照组）；通过MTT法检测各组CIK、NK细胞在不同效靶比下对K562的杀伤率，计算各组的总杀伤单位。结果：经过21天培养A组体系中CIK＋NK细胞的比例平均超过60％，B组CIK＋NK细胞的比例平均超过70％，而C组约为50％；各组扩增的CB-CIK／NK细胞同组间在效靶比20：1时对K562的杀伤率均显著高于10：1（P〈0．01）。在不同效靶比下A组CIK／NK细胞对K562的杀伤率显著高于B和C组（P〈0．01）；C组CIK／NK细胞对K562的杀伤率稍高于B组，但两组间对K562的杀伤率无显著性差异。A组总杀伤单位显著高于C组，与B组无显著差异。结论：不同细胞因子组合扩增的脐血CIK、NK细胞对人K562细胞株的杀伤活性有差异，考虑到对效应细胞的扩增效果，B组为具有最佳杀伤活性的脐血CIK、NK细胞培养体系，其中的SCF、FLT3L对CIK／NK细胞诱导有协同效果。     

脐血CIK细胞诱导K562细胞的凋亡作用

探讨脐血CIK细胞对K562细胞的杀伤活性及诱导凋亡作用。通过第1天在脐血淋巴细胞中加入IFN-r，第2天再加入IL-2和CD3单抗进行细胞培养获得多种细胞因子诱导的杀伤细胞(Cytokines induced killer cells)即脐血CIK细胞。用MTT法测定其抗肿瘤活性，原位末端标记法进行凋亡分析，台盼蓝拒染法计数细胞。脐血CIK细胞及培养上清抗K562活性明显优于CD3AK细胞，短期培养后其增殖活性低于CD3AK细胞；但是加入G-CSF可以提高CIK细胞的增殖活性，且CIK细胞的抗K562活性仍高于CD3AK细胞。CIK细胞诱导K562细胞凋亡率大于CD3AK细胞，G-CSF能部分抑制CIK细胞诱导的K562细胞的凋亡。脐血CIK细胞是一种有效的杀伤活化细胞，脐血在多种细胞因子适当组合的诱导下可提高杀瘤活性。     

Suppression of monocyte mediated anti U562 cytotoxicity by soluble factors in the serum of cancer patients and in the supernatant of K562 cell culture

Anti U562 cytotoxicity of monocytes isolated from blood of patients with alimentary tract cancer was shown to be depressed compared to monocytes of healthy individuals. A serum factor was demonstrated in the patients which depressed the cytotoxicity of healthy donor monocytes. Such suppressive factor was also produced by the K562 cell line and its production was blocked by indomethacin, a prostaglandin synthesis inhibitor, as well as by thymus preparations TFX and Thymex L.