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以下是current protocol in pharmacology中的步骤，没法贴附件，可通过邮件发给您！
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1. Serially dilute the 1.0 mM L-carnosine (or other test compound) with HBSS to prepare 200 μl each of five solutions at 100, 10, 5, 2.5, and 1 μM.
2. Add 100 μl of 0.05 N HCl to each standard solution.
3. Using a Pasteur pipet connected to a vacuum pump, aspirate the medium from both the apical (donor) and basolateral (receiver) sides of three Transwells of cells grown for 21 to 28 days. Wash the cell monolayers by adding 1.5 ml and 2.6 ml of 37°C HBSS to the apical and basolateral compartments, respectively, and then aspirating the HBSS from both compartments. Repeat.
4. Add fresh 1.5-ml and 2.6-ml aliquots of HBSS to the Transwells and incubate 30 min in a shaking water bath (37°C, 55 rpm).
5. Aspirate HBSS from both sides and add 2.6 ml of 37°C HBSS to the receiver side. Add 1.5 ml of 1.0 mM L-carnosine at 37°C to the donor side. Place the Transwells in the shaking water bath.
6. Remove 20-μl samples from the donor side at 0, 60, and 120 min, and mix each with 280 μl of 0.01 N HCl. Remove 200-μl samples from the receiver side at 15, 30, 45, 60, 90, and 120 min, and mix each with 100 μl of 0.05 N HCl. Replenish the receiver side with the same volume (200 μl) of 37°C HBSS after each sampling.
7. Determine the amount of test compound in the transport experiment samples and the standards (step 2) by HPLC (injection volume = 140 μl). Prepare a standard curve and determine the concentrations of the samples from the transport experiment by comparing their peak areas with those obtained from the standard curve.
8. Plot concentration versus time for the samples from the donor side.
9. Calculate the cumulative amount, Q (in mmol), of L-carnosine transported to the receiver side at each time point using the concentration data from step 7. In the calculations, take into account the dilution of the receiver solution (i.e., at each sampling, 200 μl solution was removed and 200 μl fresh buffer was replenished).
10. Plot the cumulative amount transported (Q) versus time (see Fig. 7.2.2) and determine the linear appearance rate (slope = ΔQ/Δt) of L-carnosine on the receiver side.
11. Calculate the Papp (in cm/sec) according to the following equation: Papp =
(ΔQ/Δt)/(ACo), where ΔQ/Δt is the linear appearance rate of the compound on the receiver side (in mmol/sec) determined in the last step (convert the time unit
accordingly); A is the surface area of the cell monolayers (in cm2; 4.71 cm2 for
Transwells 24 mm in diameter); and C0 is the initial concentration of the compound on the donor side (in mmol/cm3). See Table 7.2.1 for original data and calculated results; see Anticipated Results for interpretation of the results. |
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