Expression of the Gag.eYFP.late fusion protein and the release deficiency of this construct was confirmed by immunoblot analysis of cell lysate and pelleted tissue culture supernatant.
Subsequently, the pelleted tissue pieces were resuspended in 15 ml Hank's Buffered Salt Solution (HBSS; PAA) with 0.1%% (w/v) collagenase II (Invitrogen) and incubated at 37°C while stirring for 1 to 1.5 h (kidney) or 2 h (ear).
Briefly, samples were immersed in xylene for 5 minutes, centrifuged to pellet the tissue and, to enable removal of the xylene, washed twice in ethanol (1×85% and 1×100%), then allowed to air dry at 75°C for 5 minutes.
Centrifuge at 1,200 rpm for 2 min to pellet resuspended tissue.
Samples consisting of calibrators, Quality control (QC), cell pellet or tissue homogenate were spiked with 2 nm of d4-serotonin.
To 500 μL of calibrator, cell pellet or tissue homogenate 20 μL of d4-5-HT solution (100 μM) was added.
Chloroform (300 μl) was subsequently added to the samples and the tubes vortexed vigorously before centrifugation to pellet the tissue residue.
The resuspended cell pellet or tissue was vortexed for 10 seconds, then incubated on ice for 20 min, and then centrifuged at 20,000 g for 20 min.
