Evaluation of anchorage-independent proliferation in tumorigenic cells using the redox dye alamarBlue.
Evaluation of anchorage-independent proliferation in tumorigenic cells using the redox dye alamarBlue.
复制标题
使用氧化还原染料 alamarBlue 评估致瘤细胞的锚定非依赖性增殖。
DOI：
10.2144/96215bm05
复制
发表时间：
1996
期刊：
影响因子：
2.7
通讯作者：
E. Wickstrom
中科院分区：
文献类型：
--
作者：
Gary D. Gray;E. Wickstrom
细胞增殖是细胞生物学研究的一个关键焦点，涉及发育、衰老、免疫激活、肿瘤发生和基本细胞过程，如信号转导、DNA转录和DNA复制。已经描述了用于评估培养物中细胞增殖的多种技术，包括通过手动或自动化手段进行的直接细胞计数、通过流式细胞术进行的细胞周期分析（8）、将标记或修饰的核苷酸掺入DNA（2、9、12）、通过DNA结合荧光团测量总DNA（3、14）、通过流式细胞术进行的细胞周期分析（15）、通过流式细胞术进行的细胞周期分析（16）、通过流式细胞术进行的细胞周期分析（17）、通过流式细胞术进行的细胞周期分析（18）、通过流式细胞术进行的细胞周期分析（19）、通过流式细胞术进行的细胞周期分析（13）和通过吸收或荧光光谱法检测代谢还原的染料（10，14）。这些不同的方法在不同程度上平衡了灵敏度、可靠性、成本、仪器要求和可加工性，但所有这些方法都是针对评估在悬浮液中生长或作为附着单层生长的细胞。与肿瘤发生相关的增殖最好在锚定非依赖性生长条件下进行检查，细胞固定在软琼脂或琼脂糖中（6）。在这些条件下的增殖评价仅限于克隆集落的直接人工计数或自动图像分析。前一种方法费时，不利于分析大量样品，而后一种方法需要昂贵的专门设备。在这里，我们报告了使用alamarBlue染色剂（AlamarBiosciences，萨克拉门托，CA，USA）来评价固定在琼脂糖中的细胞的增殖。AlamarBlue以前曾用于监测悬浮生长或作为附着单层生长的细胞的增殖（2、4、11）。当添加到培养基中时，染料被细胞线粒体酶还原，产生与细胞数量成正比的可溶性产物。该产品具有较低的峰值吸光度值和显著增强的荧光（11），其可以在细胞培养基中通过光谱法测量而无需进一步处理。由于该方法不需要处理用于光谱测量的培养基或细胞，因此它很容易适用于固定在琼脂糖或软琼脂中的细胞。实验中使用了三种致瘤细胞系。将来源于人膀胱癌的EJ细胞（1，5）在37 ℃、5%CO2下在补充有10%热灭活胎牛血清、青霉素（100 U/mL）、链霉素（100 μg/mL）和2 mM谷氨酰胺的Dulbecco改良Eagle培养基（DMEM）中维持。将来源于非小细胞肺癌的NCI-H157细胞（CRL-5802; ATCC，Rockville，MD，USA）和来源于卵巢腺癌的SKOV 3细胞（HTB 77 ATCC）类似地维持在如上所述补充的RPMI-1640培养基中。实验使用35-mm培养皿或在一种情况下使用24孔板进行。用初始1 mL的0.45%琼脂糖培养基制备培养皿。然后在40°C下将胰蛋白酶化的细胞与0.25%琼脂糖/培养基溶液混合至5-20 × 106个细胞/mL的浓度，并将1 mL该细胞混合物添加至每个平板的培养皿顶部。在该细胞-琼脂糖层固化后，将0.5 mL培养基覆盖在琼脂糖上。以相似的方式制备微孔板（24孔），不同之处在于两个琼脂糖层的体积为250 μL，培养基覆盖物的体积为200 μL。将细胞在37°C、5%C02的加湿培养箱中生长14-21天，每4天更换培养基覆盖物。通过单个菌落的显微镜计数（>200 μm）或通过添加alamarBlue染料后的光谱法评价增殖。对于使用染料进行的评价，用在培养基中稀释的alamarBlue（1：2用于35 mm培养皿，1：3用于24孔板）替换培养基覆盖层，使每个板的总染料浓度约为10%（vol/vol）。将细胞在37°C和5% CO2下孵育18-24 h，直至明显的颜色变化表明存在足量的还原染料以进行充分定量。对于35 mm培养皿，在不同时间点从每个平板中取出200 μL等份试样，置于96孔微孔板中，并使用595 nm作为参比波长（生产商说明书），通过570 nm处的吸光度进行分析。对于24孔板，使用激发波长为560 nm和发射波长为590 nm的荧光板读数器（制造商说明书）直接从板上进行荧光测量。在一个实验中，将EJ细胞以不同浓度接种，2-3周后，在添加alamarBlue后检查它们的菌落计数和吸光度（图1）。手动菌落计数和alamarBlue光谱法均显示出作为细胞平板浓度的函数的等效增加，表明染料程序提供了与这些条件下的菌落计数一样准确的锚定非依赖性增殖的测量。用另外两种致瘤性人细胞系（H157和SK-0 V3）获得了类似的结果。在第二系列实验中，固定化的EJ细胞在不同血清浓度存在下生长，这是一种产生不同增殖率的程序（7），并且在不同血清浓度下，EJ细胞的相对增殖率（7）是不同的。
Cell proliferation is a key focus of research in cell biology, relating as it does to development, aging, immune activation, tumorigenesis and basic cellular processes such as signal transduction, DNA transcription and DNA replication. A wide variety of techniques for evaluating cell proliferation in culture have been described including direct cell counting by manual or automated means, cell cycle analysis by flow cytometry (8), incorporation of labeled or modified nucleotides into DNA (2,9,12), measurement of total DNA by DNA-binding fluorophores (3,13) and the detection of metabolically reduced dyes by absorbance or fluorescence spectroscopy (10,14). These various methods balance sensitivity, reliability, cost, instrumentation requirements and processibility in varying degrees, but all are directed at evaluating cells growing in suspension or as an attached monolayer. Proliferation relating to tumorigenesis may be best examined under conditions of anchorage-independent growth, with cells immobilized in soft agar or agarose (6). Evaluation of proliferation under these conditions has been limited to either direct manual counting of clonal colonies or automated image analysis. The former method is time-consuming and not conducive to the analysis of a large number of samples, whereas the latter requires expensive, specialized equipment. Here, we report the use of alamarBlue dye (Alamar Biosciences, Sacramento, CA, USA) to evaluate the proliferation of cells immobilized in agarose. AlamarBlue has previously been used to monitor proliferation in cells growing in suspension or as an attached monolayer (2,4,11). When added to culture medium, the dye is reduced by cellular mitochondrial enzymes, yielding a soluble product directly proportional to cell number. The product has a lower peak absorbance value and a substantially enhanced fluorescence (11), which can be measured spectroscopically in cell medium without further processing. Because the procedure does not require processing of either the culture medium or cells for the spectroscopic measurements, it is easily adaptable for use with cells immobilized in agarose or soft agar. Three tumorigenic cell lines were used in the experiments. EJ cells derived from a human bladder carcinoma (1,5) were maintained at 37°C under 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and 2 mM glutamine. NCI-H157 cells derived from a nonsmall cell lung carcinoma (CRL-5802; ATCC, Rockville, MD, USA) and SKOV3 cells derived from an ovarian adenocarcinoma (HTB 77 ATCC) were similarly maintained in RPMI-1640 medium supplemented as described above. Experiments were conducted using either 35-mm petri dishes or, in one case, 24-well plates. The petri dishes were prepared with an initial 1-mL underlay of 0.45% agarose in medium. Trypsinized cells were then mixed with a 0.25% agarose/medium solution at 40°C to a concentration of 5–20 × 106 cells/mL, and 1 mL of this cell mixture was added to each plate on top of the underlay. Following solidification of this cell-agarose layer, an overlay of 0.5 mL medium was placed over the agarose. Microwell plates (24-well) were prepared in a similar manner, except that the volume of the two agarose layers was 250 μL, and the volume of the medium overlay was 200 μL. Cells were grown for 14–21 days in a humidified incubator at 37°C under 5% CO2, with medium overlays replaced every 4 days. Proliferation was evaluated either by the microscopic counting of individual colonies (>200 μm) or by spectroscopy following addition of alamarBlue dye. For evaluations using the dye, the medium overlay was replaced with alamarBlue diluted in medium (1:2 for use with the 35-mm dishes and 1:3 for use with the 24-well plates), yielding an overall dye concentration for each plate of approximately 10% (vol/vol). The cells were incubated for 18–24 h at 37°C under 5% CO2 until obvious color changes indicated the presence of sufficient amounts of reduced dye for adequate quantitation. For the 35-mm dishes, 200-μL aliquots were removed from each plate at different time points, placed in a 96-well microwell plate and analyzed by light absorbance at 570 nm using 595 nm as the reference wavelength (manufacturer’s instructions). For the 24-well plate, fluorescence measurements were taken directly from the plate using a fluorescence plate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm (manufacturer’s instructions). In one experiment, EJ cells were plated at varying concentrations, and after 2–3 weeks, they were examined for both colony counts and absorbance following addition of alamarBlue (Figure 1). Both manual colony counts and alamarBlue spectroscopy demonstrated equivalent increases as a function of cell plating concentration, indicating that the dye procedure provides as accurate a measurement of anchorageindependent proliferation as colony counts under these conditions. Similar results were obtained with two other tumorigenic human cell lines (H157 and SK-OV3) In a second series of experiments, immobilized EJ cells were grown in the presence of varying serum concentrations, a procedure that produces varying proliferation rates (7), and the relia-
影响因子：
2.9
作者：
R. Rago;J. Mitchen;G. Wilding
通讯作者：
R. Rago;J. Mitchen;G. Wilding
DOI：
10.1089/ard.1992.2.3
发表时间：
1992
期刊：
Antisense research and development
影响因子：
--
作者：
Kashani-Sabet,M;Funato,T;Tone,T;Jiao,L;Wang,W;Yoshida,E;Kashfinn,BI;Shitara,T;Wu,AM;Moreno,JG
通讯作者：
Moreno,JG