TransFlex MDCK-hBCRP Efflux Assay User Guide

Transcription

1 TransFlex MDCK-hBCRP Efflux Assay User Guide VERSION 1.1 Items included in the shipment: TransFlex assay system: Includes a 96-well insert plate containing polarized MDCK-II cells transfected with either a human BCRP (ABCG2) expression vector or a control vector. The system also includes a plate lid and bottom tray. Note: Transporter-expressing cells and control cells are included on the same plate. See the plate map enclosed with each TransFlex product for illustration. Two 96-well basal plates Items included in the TransFlex MDCK Starter Kit (sold separately; Cat. No. TFlex- Start-1): 8-channel aspirating manifold (Wheaton, Cat. No ) Note: The manifold is highly recommended for aspirating fluid during each wash step. The manifold is customized to allow maximal removal of fluid from the insert wells, with no damage to the cell monolayers. See video at optiviabio.com/tutorial for demonstration. Other aspiration apparatus may be used only after being tested by the customer. 8 wash trays (Millipore, Cat. No. MAMCS0110; used to wash the bottom of the 96-well insert plate) The following items are required but not supplied: Sterile CO2 cell culture incubator, set at 37 C, 5% CO2 Regular/non-sterile incubator, set at 37 C Orbital Shaker (VWR, Cat. No or similar), to be placed in a regular incubator 1x Hanks Balanced Salt Solution with Ca 2+ and Mg 2+ (HBSS; Corning, Cat. No CM or equivalent) 1x Phosphate Buffered Saline with Ca 2+ and Mg 2+ (PBS; Corning, Cat. No CV or equivalent) DMSO (solvent/vehicle control) Probe substrate: [ 3 H]-prazosin (Perkin Elmer, Cat. No. NET823 or equivalent; for radiometric analysis), or prazosin (CAS No ; for LC/MS analysis) Reference inhibitor: Ko143 (CAS No ) 1.2 ml 12-channel electronic pipette (Mettler Toledo, Cat. No. EDP3-plus μl or similar) 200 µl 8- or 12-channel manual pipette (Mettler Toledo, Cat. No. L8-200XLS+ or similar) 100 ml disposable reagent reservoir Vacuum aspiration system 2 ml 96-well dosing block, preferably glass-coated to minimize potential nonspecific binding (NSB) of compounds (Thermo Scientific, Cat. No P308 or equivalent) WorkWipes Series 30 Wipers (absorbent wipes for drying the bottom of the 96-well insert; New Pig Corp, Cat. No. WIP630 or similar) 1

2 Assay Conditions and Specifications Table I. Summary of Assay Conditions Probe substrate and test concentration: prazosin at 2 µm Reference inhibitor and test concentration: Ko143 at 1 µm Vehicle Control: 0.5% DMSO Transcellular transport direction: Basal to apical (B>A) & apical to basal (A>B) Volume of assay solutions: Basal/Bottom: 300 µl, Apical/Top: 150 µl Preincubation solution: Substrate study: HBSS, with Ca 2+ and Mg 2+ Inhibition study: HBSS, with Ca 2+ and Mg 2+, containing vehicle control or test articles Preincubation time: 30 minutes Efflux assay buffer: HBSS, with Ca 2+ and Mg 2+ Efflux assay time: 90 minutes Monolayer surface area: 0.27 cm 2 Table II. Typical Assay Activities Under the Specified Assay Conditions Average Efflux Ratio (Papp,B>A/Papp,A>B) of probe substrate in MDCK-hBCRP cells: 5 % inhibition by reference inhibitor: 73.5% Paracellular leakage (measured by mannitol): 2%/hour Typical CV of triplicates: 30% General Principles and Terminology of Bi-directional Efflux Assays Efflux assays involve the measurement of transcellular drug transport in both the basal-to-apical (B>A) and the apical-to-basal (A>B) directions. The wells of the 96-well basal plate constitute the basal compartments, while the wells of the insert plate constitute the apical compartments. In substrate assays, B>A transport is assessed by adding a compound to one of the basal compartments (i.e., the dosing compartment) and measuring the amount of compound appearing in the apical compartment (i.e., the receiving compartment). Alternatively, A>B transport requires adding the compound to the apical compartment/insert well (now the dosing compartment), and measuring the amount of compound appearing in the basal well (now the receiving compartment). The efflux ratio (defined as the ratio of apparent permeabilities, Papp,B>A/Papp,A>B) is commonly used to quantify the vectorial transport caused by transporter-mediated active efflux of a substrate. In inhibition assays, the probe substrate is added as described above, while the compound to be tested as a potential inhibitor is typically added to both the apical and basal compartments during the preincubation and the transcellular assay. TransFlex MDCK-hBCRP Efflux Assay User Guide, v.1.1 2

3 Equipment Preparation 1. Have a sterile CO2 incubator ready (set at 37 C, 5% CO2) at least one day before receiving the TransFlex product. 2. At least one day before conducting the transport assay, prepare a regular incubator by setting the temperature to 37 C, and placing a beaker containing at least 200 ml dh2o inside to humidify the interior. Assay Protocol 1. Unpack the TransFlex assay system Remove the TransFlex product from the sealed bag Carefully wipe the product exterior with 70% isopropyl alcohol and place in a 37 C, 5% CO2 sterile cell culture incubator. It is not necessary to change the medium. Allow the cells to incubate for hours before assaying as described below. Note: The cells are intended to be assayed within the time period specified above. Activity cannot be guaranteed if the assay is performed outside of the recommended time period. 2. Prepare the wash buffer. Warm HBSS (~200 ml/plate) to 37 C. 3. Prepare the preincubation and assay solutions Calculate the total volume of dosing solution required for each test condition. For basal-to-apical (B>A) transport, use 300 µl 1x dosing solution per assay well; for apical-to-basal (A>B) transport, use 150 µl 1x dosing solution per assay well. Include at least 10% more volume than you expect to need, in order to accommodate pipetting errors. For example, for one test condition involving 3 triplicate transporter wells and 3 triplicate control wells, we recommend making at least 3 ml of the 1x final dosing solution To reduce pipetting/handling errors, we recommend placing the dosing and receiving solutions in a 96- well dosing block first, and then using a high-precision, 8-channel pipette to transfer the solutions, one column a time, to the appropriate assay plates. Assuming a standard transporter plate configuration (with 3 transporter wells and 3 control wells for each transcellular assay), the liquid in one column of the dosing block should be sufficient to fill one half of a 96-well basal or insert plate (e.g., columns 1 6, which are typically used for B>A transport, or columns 7 12, which are typically used for A>B transport) Prepare the solutions and basal plate for the substrate assay as follows: A. Receiving Solution: Prepare 25 ml of an HBSS solution containing the vehicle control at its final test concentration (0.5% DMSO); this will serve as the receiving solution for one whole plate of substrate assays. B. Probe Substrate Dosing Solution: In a glass vial, prepare at least 3 ml of an HBSS solution containing the probe substrate and vehicle at their final test concentrations (e.g., 2 µm prazosin and 0.5% DMSO). C. Test Article Dosing Solution(s): For each test article, use a glass vial to prepare at least 3 ml of an HBSS solution containing the test article at its final test concentration and 0.5% DMSO. D. Prepare the substrate-assay dosing block: For the B>A assay, transfer 2 ml of each of the above dosing solutions to the appropriate wells in one column of a 2 ml, 96-well dosing block, then transfer 1 ml of the receiving solution to each of the wells in another column. For the A>B assay, transfer 1 ml of each of the above dosing solutions to the appropriate wells in one column of the dosing block, then transfer 2 ml of the receiving solution to each of the wells in another column. Warm the solutions to 37 C by placing the covered dosing block into an incubator for at least 20 minutes. TransFlex MDCK-hBCRP Efflux Assay User Guide, v.1.1 3

4 E. Approximately 10 minutes before starting the transport assay (Step 5), use a high-precision, 8- channel pipette to transfer 300 µl of the following solutions, one column at a time, from the dosing block to the appropriate wells of one 96-well basal plate: (i) add the pre-warmed dosing solutions to the wells corresponding to the B>A assays; and (ii) add the pre-warmed receiving solution to the wells corresponding to the A>B assays. Place the covered basal plate and the assay dosing block into a 37 C incubator for later use Prepare the solutions and basal plates for the inhibition assay as follows: Prepare the preincubation solutions and basal preincubation plate. A. Vehicle Control: Prepare at least 6 ml of an HBSS solution containing the vehicle control at its final test concentration (0.5% DMSO). B. Reference Inhibitor: In a glass vial, prepare at least 6 ml of an HBSS solution containing 1 µm Ko143 and 0.5% DMSO. C. Test Article(s): For each test article, use a glass vial to prepare at least 6 ml of an HBSS solution containing the test article at its final test concentration and 0.5% DMSO. D. At least 20 minutes before preincubation (Step 4.7), use a high-precision pipette to add 300 µl of these solutions to the appropriate wells of one of the 96-well basal plates provided. To avoid confusion, this plate will be referred to as the basal preincubation plate, and should be labeled appropriately. Cover the basal preincubation plate and place it in a 37 C incubator. Notes: 1) For ergonomic and error-free handling, the use of a high-precision, electronic pipette with a repetitive pipetting/multi-dispense mode is highly recommended. 2) In the standard plate configuration and experimental design described in 3.2, each row of the basal preincubation plate should contain the same solution, despite the fact that half of each row is for B>A transport, and half is for A>B transport. E. Warm the remaining preincubation solutions to 37 C for later use by placing the vials on a heat block or in an incubator Prepare the inhibition assay solutions and basal assay plate. A. Vehicle Control: For the dosing solution, prepare at least 3 ml of an HBSS solution containing the probe substrate and vehicle control at their final test concentrations (e.g., 2 µm prazosin and 0.5% DMSO). For the receiving solution, prepare at least 3 ml of an HBSS solution containing the vehicle control at its final test concentration (e.g., 0.5% DMSO). B. Reference Inhibitor: For the dosing solution, prepare at least 3 ml of an HBSS solution containing 2 µm prazosin, 1 µm Ko143 and 0.5% DMSO. For the receiving solution, prepare at least 3 ml of an HBSS solution containing 1 µm Ko143 and 0.5% DMSO. C. Test Article(s): For each test article: (i) prepare a dosing solution of at least 3 ml HBSS containing the test article at its final test concentration, 2 µm prazosin and 0.5% DMSO; (ii) prepare a receiving solution of at least 3 ml HBSS containing the test article at its final test concentration and 0.5% DMSO. D. Prepare the inhibition-assay dosing block: For the B>A assays, transfer 2 ml of each of the above dosing solutions to the appropriate wells in one column of a 96-well dosing block, then transfer 1 ml of each of the corresponding receiving solutions to the appropriate wells in another column. For the A>B assay, transfer 1 ml of each of the above dosing solutions into the appropriate wells in one column of the dosing block, then transfer 2 ml of each of the corresponding receiving solutions into the appropriate wells in another column. Warm the solutions to 37 C by placing the covered dosing block into an incubator for at least 20 minutes. TransFlex MDCK-hBCRP Efflux Assay User Guide, v.1.1 4

5 E. Approximately 10 minutes before starting the transport assay (Step 5), use a highprecision, 8-channel pipette to transfer 300 µl of the following solutions, one column at a time, from the inhibition-assay dosing block to the appropriate wells of one of the 96-well basal plates provided: (i) add the pre-warmed dosing solutions (containing the probe substrate) to the wells corresponding to the B>A assays; and (ii) add the pre-warmed receiving solutions (without the probe substrate) to the wells corresponding to the A>B assays. To avoid confusion, this plate will be referred to as the basal assay plate and should be labeled appropriately. Place the covered basal assay plate and inhibition-assay dosing block into a 37 C incubator for later use. 4. Wash and pre-incubate the cells. Note: The following steps do not require sterile conditions. However, it is important to handle the 96- well insert plate with care to prevent damage to the cell monolayers. To minimize disturbance to the cell monolayers, it is also important to avoid pipetting solutions directly onto the cells in the insert wells; instead, solutions should be dispensed with pipette tips aimed at the walls of the wells Place ~80 ml of 37 C HBSS into a disposable pipetting reservoir Prepare 3 separate wash trays by placing ~25 ml of 37 C HBSS into each Carefully separate the insert plate from the bottom tray containing shipping medium Wash the bottom of the insert plate by placing it into the first wash tray. Use the 8-channel aspirating manifold to remove the liquid from the wells of the insert plate, then add 150 µl of 37 C HBSS to each insert well. Repeat the HBSS wash two more times (using a fresh wash tray each time). Note: While using the aspirating manifold to remove liquid from the wells: i) Hold the manifold straight up-and-down (not at an angle) against the walls of the wells. ii) Once the manifold has been fully inserted into the wells, hold it still do not move it around After the last wash, completely aspirate the HBSS from all of the insert wells. Dry the bottom of the insert plate by blotting on WorkWipes. Note: After blotting, visually inspect the underside of the insert plate to make sure there is no residual liquid between the insert wells. If liquid is present, it can be removed by gently flicking and re-blotting the insert plate Remove the shipping medium from the bottom tray that came with the TransFlex system. Rinse the tray once with HBSS and aspirate the liquid. Dry the tray and use in Step Preincubate the cells. A. Substrate assay: Add 150 µl warm HBSS to each well of the washed insert plate, and 25 ml warm HBSS to the bottom tray. Place the insert plate into the bottom tray, cover the insert plate with the lid, and incubate the resulting plate assembly on an orbital shaker (at 60 rpm) in a 37 C incubator for 30 minutes. B. Inhibition assay: Place the insert plate into the dry bottom tray and add 150 µl of the warmed preincubation solutions (from Step E) into the appropriate insert wells. Remove the basal preincubation plate (Step D) from the incubator. Place the insert plate into the basal preincubation plate, cover the insert plate with the lid, and incubate the plate assembly on an orbital shaker (at 60 rpm) in a 37 C incubator for 30 minutes. Note: To prevent bubbles from becoming trapped between the basal plate and the insert plate: Place the basal preincubation plate/bottom tray on a level surface. Align the two plates, then tilt the insert plate ~20 30 degrees and slowly lower it into the basal plate. TransFlex MDCK-hBCRP Efflux Assay User Guide, v.1.1 5

6 5. Perform the efflux assay Completely aspirate the liquid from all of the insert wells, and dry the bottom of the insert plate by blotting on WorkWipes. After blotting, visually inspect the underside of the insert plate and remove any residual liquid that may be present Place the insert plate into the dry bottom tray, then use a high-precision, 8-channel pipette to quickly transfer 150 µl of the receiving solutions, one column at a time, from the assay dosing block (Step 3.3.D or D) to the insert wells corresponding to B>A transport; transfer 150 µl of the dosing solutions from the assay dosing block to the insert wells corresponding to A>B transport Remove the basal assay plate (Step 3.3.E or E) from the 37 C incubator and place it on the lab bench Start the transport assay by carefully placing the insert plate into the basal assay plate and covering the insert plate with the lid. Note: To prevent bubbles from becoming trapped between the assay plate and the insert plate: Place the basal assay plate on a level surface. Align the two plates, then tilt the insert plate ~20 30 degrees and slowly lower it into the basal plate Immediately place the plate assembly on an orbital shaker (set at 60 rpm) in a 37 C humidified incubator, and incubate for 90 minutes. 6. Stop the reaction and collect samples At the end of the incubation period, retrieve the plate assembly from the incubator and place it on the bench. Stop the reaction by separating the insert plate from the basal assay plate and quickly blotting the bottom of the insert plate on WorkWipes After blotting, place the insert plate into the dry bottom tray and use a high-precision, multi-channel, manual pipette to sample the desired amount of solution (up to 100 µl) from the wells of the insert plate (i.e., the apical samples), and the wells of the basal assay plate (i.e., the basal samples) Transfer the samples to an appropriate 96-well sample plate for downstream compound quantification/analysis. To facilitate downstream analysis, it is recommended that the samples from the receiving compartments be grouped together and transferred to one 96-well sample plate, and the samples from the dosing compartments be grouped together and transferred to another 96-well sample plate. A. For liquid scintillation analysis: Transfer 30 µl of each assay sample to a 96-well sample plate (such as Perkin Elmer Product No , or equivalent). Add 200 µl scintillation fluid (such as Ultima Gold XR cocktail; Perkin Elmer, Product No ) to each well, seal and vortex the plate, then read on a scintillation counter. B. For LC/MS analysis: Transfer 30 µl of each assay sample to a low-binding sample plate with each well pre-loaded with 30 µl of an internal standard. Gently pipette to mix. Seal the sample plate and proceed with LC/MS analysis, or keep the plate at 80 C for later use. C. For fluorescence- or luminescence-based analysis: Transfer 30 µl of each assay sample to a low-background sample plate. Follow your preferred fluorescence- or luminescence-based assay protocol to analyze the samples. TransFlex MDCK-hBCRP Efflux Assay User Guide, v.1.1 6