药物转运的细胞相关测定
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概述
规格
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 |
|---|---|
| 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. |
| Can you use all 96 wells for the drug permeability assay in the MultiScreen Caco-2 plate? | Typically we use all 96 wells for our assays. But as is the case for many cell based assays in 96 well plates, there may be more variability in the cell growth on the periphery of the plate. We confirm monolayer integrity by measuring the TEER on all wells prior to beginning a drug transport assay. |
| Why are the propranolol and testosterone values from Millipore’s laboratory determined on the MultiScreen Caco-2 plate lower than published values? | We have speculated that the differences we observe are related more to the cell culture and analytical conditions we employ in our laboratory because the values we observe on the standard 24 well plates are also lower than published. Because we correlate well with the 24 well data generated at the same time in our laboratory, the discrepancy from published is not a concern. |
| What kind of correlation should we expect between the MultiScreen Caco-2 plate and our current assay plates? | Provided careful optimization has been performed on the 96 well plate, the data generated should be comparable to that observed with current 24 well systems. |
| Can I grow MDCK cells on the Multiscreen Caco-2 plate? | MDCK cells are canine kidney cells that can form differentiated monolayers with tight junctions after 3 days. The permeability values for MDCK correlates well with Caco-2 and human absorption for passively absorbed compounds. In the past we have successfully demonstrated MDCK growth on our Caco-2 plate. We have discovered that cells grown for 3 days are more sensitive to fluctuations in growth conditions. It is crucial that the cell growth conditions are optimized. |
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