English to Russian Medical Medical (general)

The unbound fraction of the test compound was determined based on the protocols described below.
1)Dilute initial 10mM test article to 125μM by adding 5μL to a total volume of 395μL solvent solution (100% acetonitrile) in a 1mL 96-well plate (Waters 186002481 Milford, MA).
Ensure that the compounds are in solution.
2) Thaw frozen (rat, human, mouse, dog, and/or monkey) plasma (BIOIVT, Westbury, NY) in and warm the PBS buffer in a warm (37°C) water bath.
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Dilute the 125uM test article solutions by adding 8μL to a final volume of 992μL of plasma to make a final concentration of 1μM in a 2mL 96 well plate (Costar 3961).
Mix thoroughly.
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This spiked plasma solution is shown in figure 1.
3) Prepare a chilled ‘crash’ solution of internal standard in a solvent solution.
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Pipette 200μL of 25ng/mL solution of internal standard, CPDPX (8-Cyclopentyl-1,3-dipropylxanthine, Sigma-Aldrich, C101) in 1:1 acetonitrile/methanol solvent solution into a 1mL 96-well plate.
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Chill on ice or refrigerate at 4°C.
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This solution becomes the ‘Crash’ plate in figure 1.
4)From remaining spiked plasma, remove 50μL (T=0h) of each plasma sample and place into a crash plate which contains 200μL.
To matrix match, add 50μL of blank buffer to the crashed sample (similar to PPB samples).
Maintain remaining spiked plasma at 37˚C for 4h time point
5) Transfer 500μL of the warmed PBS buffer to the white side of the RED device (Thermo Scientific, Rockford IL, baseplate cat# 89811, insert cat# 89810) and 300 μL of the spiked plasma to the corresponding red ring side of the RED device.
6) Cover all the RED device plates with a lid and transfer them to a 37˚C incubator with 5% CO2 environment and shake at 200 rpm for 4 hours.
7)Reaction Termination after 4 hours:
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******Add 50μL of sample (plasma or buffer sample) and 50μL of the opposite blank matrix (add blank buffer to the plasma samples and blank plasma to the buffer samples) to crash plates (same as above) to the crash plate containing 200μL.
Mix crash plate thoroughly*******.