Description
Parallel Artificial Membrane Permeability Assay-BBB Kit (BA0261) (BA0261)
The Parallel Artificial Membrane Permeability Assay-BBB Kit (SKU: BA0261) is designed to aid in evaluating blood-brain barrier (BBB) membrane permeability. Membrane permeability is an important characteristic when evaluating compounds as potential drug candidates, since drugs often need to cross cell membranes to reach their target of action. The BBB is made of brain endothelial cells with tight junctions, and rapid, early screening of compounds for BBB penetration is highly desirable in drug discovery. While permeability can be evaluated by cell-based methods, these are often expensive and time-consuming; Parallel Artificial Permeability Assays (PAMPA) instead offer a quick, inexpensive means of evaluating the permeability of test compounds. This kit provides all the necessary components to run a PAMPA plate for blood-brain barrier studies, including donor and acceptor plates, dodecane, dried brain lipids and high- and low-permeability controls. The procedure is straightforward, low-cost and readily automated for high-throughput screening.
| Product Name: | Parallel Artificial Membrane Permeability Assay-BBB Kit (BA0261) |
| SKU: | BA0261 |
| Detection Method: | Parallel Artificial Membrane Permeability Assay (PAMPA); UV absorbance (200-500 nm) |
| Sample Type: | ['Test compounds'] |
| Species Reactivity: | All |
| Assay Time: | 18 hours incubation (typically 16-24 hours) |
| Kit Size: | 96 Assays |
| Equipment Required: | Microplate reader (UV/absorbance spectrum capable) |
| Storage: | Store Permeability Controls, Dodecane and Dried BBB Lipids at -20°C; store Donor Plate, Acceptor Plate and Working Tray at room temperature |
| Shelf Life: | 12 months |
| Shipping: | Room Temperature |
PAMPA kit providing all components required to run a 96-well artificial membrane permeability assay for the quantitative determination of blood-brain barrier membrane permeability.
- Convenient: includes all necessary equipment to run a PAMPA plate.
- Simple and low-cost: procedure is easy to follow and more affordable than cell-based permeability assays.
- High-throughput: readily automated as a 96-well plate assay for thousands of samples per day.
- Direct assessment of blood-brain barrier membrane permeability of test compounds.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Equilibrate all components to room temperature and briefly centrifuge tubes before opening. Prepare BBB Lipid Solution by resuspending the dried brain lipids in 600 µL dodecane, pipetting up and down (~100 times) until fully solubilised; store at -20°C and use within 6 days. |
| 2 | Prepare 10 mM test compound stock solutions in DMSO (the supplied Permeability Controls are provided as 10 mM DMSO solutions). |
| 3 | In separate tubes, prepare 500 µL of 500 µM Test Compound (25 µL 10 mM compound + 475 µL PBS pH 7.2); dilute Permeability Controls to 500 µM similarly. |
| 4 | Prepare 200 µM Equilibrium Standards for each compound and control (80 µL of 500 µM + 120 µL PBS pH 7.2), and a Blank Control (5 µL DMSO + 245 µL PBS pH 7.2); set aside for next-day analysis. |
| 5 | Add 300 µL PBS (pH 7.2) to wells in the acceptor plate. |
| 6 | With the donor plate still in its tray, add 5 µL BBB Lipid Solution in dodecane directly to the well membranes, taking care not to puncture them. |
| 7 | Add 200 µL of each 500 µM Test Compound and Permeability Control to duplicate wells of the donor plate (run all variables at least in duplicate). |
| 8 | Carefully place the donor plate into the acceptor plate wells and incubate at RT or 37°C for 18 hours (or 16-24 hours as desired). |
| 9 | Carefully remove the donor plate and collect the liquid in the acceptor plate wells (Acceptor Solution). |
| 10 | Add 100 µL of Acceptor Solution, Equilibrium Standards and Blank Control to wells of a UV plate (Cat # P96UV). |
| 11 | Read the absorbance spectrum from 200 nm to 500 nm in 10 nm intervals to determine peak absorbance; peak absorbance for the High and Low Permeability Controls are 250 nm and 270 nm respectively. |
Determine the Permeability Rate (Pe) using Pe = C × -ln(1 - ODA/ODE) cm/s, where ODA is the absorbance of the Acceptor Solution minus Blank and ODE is the absorbance of the Equilibrium Standard minus Blank. For an 18-hour incubation, C = 7.72×10^-6. For other incubation times, calculate C = (VD × VA) / ((VD + VA) × Area × time) cm/s, where Donor Volume (VD) = 0.2 cm3, Acceptor Volume (VA) = 0.3 cm3, Membrane Area = 0.24 cm2, and time = 64,800 s for 18 hours.
| Component | Quantity | Storage |
| Donor Plate | 1 Plate | Room temperature |
| Acceptor Plate | 1 Plate | Room temperature |
| Working Tray | 1 Plate | Room temperature |
| Dodecane | 1.5 mL | -20°C |
| Dried Brain Lipids | 1 Tube | -20°C |
| High Permeability Control | 120 µL | -20°C |
| Low Permeability Control | 120 µL | -20°C |
