药物转运的细胞相关测定
Millicell-96孔细胞培养嵌套板是一种专利96孔装置，设计用于支持Caco-2和其他细胞系的上皮细胞生长和分化。
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Millicell-96孔细胞培养嵌套板是一种专利96孔装置，设计用于支持Caco-2和其他细胞系的上皮细胞生长和分化。 更少
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概述
Millicell-96孔细胞培养嵌套板是一种专利96孔装置，设计用于支持Caco-2和其他细胞系的上皮细胞生长和分化。
细胞单层分化形成之后，可使用该装置测量通过细胞屏障的药物转运率。所有的实验步骤均在一个装置中完成。该测定可配置为自动完成，包括细胞接种、饲养、洗涤以及药物转运试验。
Millicell-96装置由具有96个下侧接触孔的过滤板、生长板（单孔或96 孔）、盖子以及96孔接收板组成。
- 符合ANSI/SBS的设计 — Millicell-96装置与自动液体处理系统和多道移液器兼容
- 结果明确 — 易于接触上侧和下侧
- 样品呑吐量增大 — 使用96孔系统，呑吐量比24孔系统增加4倍
- 产品形式灵活多用 — 有96孔和单孔饲养盘可供选用
性能
药物通透率与人体吸收相关
在 ArQule 公司，已使用 MultiScreen Caco-2 分析系统对表示主动、被动以及流出转运体的各种药物化合物进行了测试（有关化合物的完整列表，请参阅 Millipore 应用注释 AN1727EN00）。 将每种化合物的通透率对应其人体吸收的百分比值绘制成图。 使用 Statistica™ 软件生成一条非线性回归曲线。 合成曲线对于所有数据点表现出良好的似合，说明 MultiScreen Caco-2 分析系统可以对所有 25 种被测试的化合物的通透性属性进行正确的识别和分类。
药物的通透率与 24 孔分析系统相关
对于使用了 10 种氘标记药物的 Caco-2 药物转运率，使用 MultiScreen Caco-2 分析系统进行测量，并与构建的 24 孔系统所获得的药物转运率进行比较。 这一组数据比较了四个独立试验的结果。 R2 值为 0.99，表明两种格式的数据（24 孔分析相对于 Millipore 96 孔分析）是相关的。 有关被测试药物的完整列表，请参阅 Millipore 应用注释 AN1727EN00。
可重现的分析结果
体外基于细胞分析平台的稳定性对于可靠的分析结果至关重要。 本图显示了 MultiScreen Caco-2 分析系统中药物转运率的重现性，试验方法为：在每个被测试板的全部 96 个孔中使用经过 21 天培养的 Caco-2 细胞，并只使用一种药物：心得安 (propranolol)。 转运率由不同日期进行的 3 轮连续试验测定，每轮试验使用 4 个不同生产批次的 MultiScreen 板。 通过对板的平均值进行变量分析，可看出不同生产批次测试或不同日期板间和板内标准差的差异不显著。 经计算，总标准差为：0.3x10-5cm/sec。
规格
| 材料 | ||
| 过滤板，接收板，盖子 | 聚苯乙烯 | |
| 滤膜 | 聚碳酸酯（PC） | |
| 尺寸 | ||
| 长度 | 128 mm | |
| 宽度 | 84.8 mm | |
| 高度 | 26.2 mm | |
| 过滤面积 | 0.11 cm² | |
| 滤膜孔径 | 0.45 µm | |
| 建议的样品体积 | ||
| 过滤板 | 75 µL–100 µL | |
| 接收板 | 250 µL–300 µL | |
| 板的配置 | ||
| 槽孔类型 | 96 孔 | |
| 下侧接触孔 | 96 | |
| 槽孔阵列 | SBS | |
| 自动化试验方案 | ||
| 用下列自动化系统可以实现试验方案：Brandel、Tecan Genesis®、Zymark | ||
订货信息
Documentation
参考
| 参考概述 | 应用 |
|---|---|
| Use of ADME screening to conserve resources in drug discovery and development Weiss, A. and Joseph Machamer American Biotechnology Laboratory, January 2004: 16-17 2004 | |
| Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity. Chisato Yamasaki, Yumiko Natori, Xun-Ting Zeng, Mari Ohmura, Shinji Yamasaki, Yoshifumi Takeda and Yasuhiro Natori FEBS Letters 442 (2-3): 231-234 1999 | Cell Culture |
问与答
| 问题 | 回答 |
|---|---|
| How do you determine the optimal seeding density on the MultiScreen Caco-2 plate? | This requires optimization for each laboratory, due to the variability in Caco-2 cultures. We recommend starting with the same number of cells/cm2 used for a 24 well culture. For example if you are plating 30,000 cells/well in a 24 well plate, you would plate 11,000 cells per well for the MultiScreen Caco-2 (30,000 cells/0.3cm2)(0.11cm2)= 11,000 cells. |
| Why do we recommend addition of 75 µL of medium apically (in the filter well) and 250 µL basolaterally (in the receiver/feeder plate) for the MultiScreen Caco-2 plate? | These volumes were determined to be optimal for cell growth when feeding the cell culture every other day. In addition, with 75 µL apical, the medium level is at the same height as the basolateral volume of 250 µL. In this way we ensure that there is no positive pressure exerted on the monolayer of Caco-2 cells. |
| Do you recommend a specific way of seeding the cells on a MultiScreen Caco-2 plate? | We recommend pipetting cells at the apical notch and down the side of the well (slow speed if programmable). When seeding many plates, make sure to continually keep cells suspended. |
| Do you recommend optimal feeding conditions for cells grown on the MultiScreen Caco-2 plate? | We recommend feeding every other day (Mon, Wed, Fri.) exchanging apical and basolateral media in a specific order. The preferred order is to aspirate basolaterally first, then apically, and add fresh medium back apically first, then basolaterally. |
| Why is there a defined order for aspirating medium and addition of new medium when feeding the cells on the MultiScreen-Caco-2 plate? | When culturing epithelial cells on filters, it is desirable to limit any pressure the medium may exert which may disrupt the cell contact with the filter. Therefore, the medium in the apical compartment should always be present when medium is present in the basolateral compartment. The preferred order is to aspirate basolaterally first, then apically, and add fresh medium back apically first, then basolaterally. |
| Do you recommend any specific handling when feeding the cells on the MultiScreen-Caco-2 plate? | We recommend using an autoclavable 8-channel aspiration manifold. Sigma M2656. Drummond #3-000-093 Aspirate/feed apically using the apical notch and down the side of the well (slow speed if programmable). If you are feeding manually, feel free to separate the plates to aspirate/feed basolaterally. Due to the fact that the MultiScreen-Caco-2 plate has recessed filter wells, the membrane will not be in direct contact with any surface. The filter plate has 4 feet it stands on. |
| How much does the TEER manual electrode STX-100M cost that is recommended for use with the MultiScreen Caco-2 plate? | The STX-100M probe is $1,200 and you would order this through World Precision Instruments. You will also need a Ohm meter, which is available from Millipore, MERS 000 01. |
| What are normal TEERs (Trans epithelial electrical resistance) that are measured on the MultiScreen Caco-2 plate? | Generally, TEER values obtained are in the range of 1000-4000 ohms for a 21 day culture and 1000-3000 ohms for a 10 day culture. We consider a failing well below 1000 ohms. If you were using a 24-well plate you can estimate desired TEERs on our plate comparing the surface area. If not, you can estimate a normal range comparing lucifer yellow test results. |
| What lucifer yellow and drugs do you use on the MultiScreen Caco-2 plate? What are your test conditions? | We use lucifer yellow dipotassium salt (LY) from Sigma (L0144) concentration of 100 µg/mL in HBSS). We read LY with a Victor Plate Reader (Wallac/PE). We use tritium labeled drugs spiked with unlabeled drug and use a Microbeta reader (Wallac/PE). We usually run the LY and drug transport experiments for 2 hours, room temperature, shaking at 60 rpm. |
| Can I add lucifer yellow and drug compounds together in the same well in the MultiScreen Caco-2 plate? | We do not recommend using lucifer yellow in the same well as your test compound if you are using radioactive drugs. Lucifer yellow may interfere with scintillation cocktail and give inaccurately high counts in the beta counter. |
相关产品和应用
相关产品，按下列排序： Application Facete
| Dialysis and Filtration |
相关产品，按下列排序： Brand Facete
| Millicell® |
种类
| Life Science Research > Cell Culture and Systems > Cell Culture Flasks, Plates, & Slides > Multiwell Plates |
| 材料 | ||
| 过滤板，接收板，盖子 | 聚苯乙烯 | |
| 滤膜 | 聚碳酸酯（PC） | |
| 尺寸 | ||
| 长度 | 128 mm | |
| 宽度 | 84.8 mm | |
| 高度 | 26.2 mm | |
| 过滤面积 | 0.11 cm² | |
| 滤膜孔径 | 0.45 µm | |
| 建议的样品体积 | ||
| 过滤板 | 75 µL–100 µL | |
| 接收板 | 250 µL–300 µL | |
| 板的配置 | ||
| 槽孔类型 | 96 孔 | |
| 下侧接触孔 | 96 | |
| 槽孔阵列 | SBS | |
| 自动化试验方案 | ||
| 用下列自动化系统可以实现试验方案：Brandel、Tecan Genesis®、Zymark | ||