Cell-based Assays for Drug Transport
Millicell-96 cell culture insert plate is a patented 96-well device designed to support epithelial cell growth and differentiation of Caco-2 and other cell lines.
More
Millicell-96 cell culture insert plate is a patented 96-well device designed to support epithelial cell growth and differentiation of Caco-2 and other cell lines. Less
More>>
Less<<
Overview
Millicell-96 cell culture insert plate is a patented 96-well device designed to support epithelial cell growth and differentiation of Caco-2 and other cell lines.
After the formation of a differentiated cell monolayer, the device can be used to measure the rate of drug transport across the cell barrier. All procedures are designed to be carried out in a single device. The assay can be performed using automation for cell seeding, cell feeding, washing and drug transport experiments.
The Millicell-96 device consists of a filter plate with 96 basolateral access holes, growth plate (single well and 96-well available), lid and 96-well receiver plate.
- ANSI/SBS-compliant design – the Millicell-96 device is compatible with automated liquid handling systems and span pipettes
- Results not compromised – ease of access to both apical and basolateral sides is assured
- Increased sample throughput – using the 96-well system, throughput is increased by four compared to 24-well systems
- Versatile format – 96- and single-well feeder trays available
Performance
Drug Permeability Rates Correlate to Human Absorption
Drug compounds representing active, passive and efflux transporters were tested at ArQule, Inc. using the Millicell-96 assay system (for a complete list of compounds, please click on the Supporting Documents tab and see application note: MultiScreen Caco-2 --Drug Transport Assembly in a 96-well system). Each compound’s permeability rate was plotted against their percent human absorption values. A nonlinear regression curve was generated using Statistica™ software. The resultant curve provided a good fit to all of the data points to demonstrate that the Millicell-96 assay system could correctly identify and classify the permeability properties of all 25 compounds tested.
Drug Permeability Rates Correlate with 24-well Assay Systems
Caco-2 drug transport rates using 10 tritium labeled drugs were measured using the Millicell-96 assay system and compared to the drug transport rates obtained from an established 24-well system. This set compares the results from four separate experiments. The R2 value of 0.99 suggests that the data between the two formats (24-well assay vs. Millipore 96-well) correlate. For a complete listing of the drugs tested, refer to Millipore Application Note AN1727EN00.
Reproducible Assay Results
Low variability of in vitro cell-based assay platforms is critical for reliable assay results. This figure examines the reproducibility of drug transport rates in Millicell-96 assay system using a 21-day culture of Caco-2 cells and a single drug, propranolol, in all 96-wells of every plate tested. Transport rates were determined in 3 experimental runs performed on different days, each run consisting of 4 different production lots of Millicell-96 plates. Analysis of variance on the plate average, inter-plate and intra-plate standard deviations does not vary significantly among the production lot tests or days. The total standard deviation is calculated to be 0.3 x 10-5 cm/sec.
Specifications
| Materials | ||
| Filter Plate, Receiver Plate, Lid | Polystyrene | |
| Membrane | Polycarbonate (PC) | |
| Dimensions | ||
| Length | 128 mm | |
| Width | 84.8 mm | |
| Height | 26.2 mm | |
| Filtration Area | 0.11 cm² | |
| Membrane Pore Size | 0.45 µm | |
| Recommended Sample Volume | ||
| Filter Plate | 75 µL–100 µL | |
| Receiver Plate | 250 µL–300 µL | |
| Plate Configuration | ||
| Well Type | 96-well | |
| Basolateral Access Holes | 96 | |
| Well Array | SBS | |
| Automation Protocols | ||
| Protocols exist for the the following automation systems: Brandel, Tecan Genesis®, Zymark | ||
Ordering Information
Documentation
References
| Reference overview | Application |
|---|---|
| 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 |
FAQ
| Question | Answer |
|---|---|
| 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. |
Related Products & Applications
Related Products By: Application Facete
| Dialysis and Filtration |
Related Products By: Brand Facete
| Millicell® |
Categories
| Life Science Research > Cell Culture and Systems > Cell Culture Flasks, Plates, & Slides > Multiwell Plates |
| Materials | ||
| Filter Plate, Receiver Plate, Lid | Polystyrene | |
| Membrane | Polycarbonate (PC) | |
| Dimensions | ||
| Length | 128 mm | |
| Width | 84.8 mm | |
| Height | 26.2 mm | |
| Filtration Area | 0.11 cm² | |
| Membrane Pore Size | 0.45 µm | |
| Recommended Sample Volume | ||
| Filter Plate | 75 µL–100 µL | |
| Receiver Plate | 250 µL–300 µL | |
| Plate Configuration | ||
| Well Type | 96-well | |
| Basolateral Access Holes | 96 | |
| Well Array | SBS | |
| Automation Protocols | ||
| Protocols exist for the the following automation systems: Brandel, Tecan Genesis®, Zymark | ||