Bromododecane

2X10 6 T cells were suspended in 200ul PBS containing permeant putrescine (100 μM) and [1,4- 14 C]-putrescine dihydrochloride (0.2 uCi, ARC 0245) and incubated at 37°C for 10 minutes (within the established linear phase of uptake). The reaction was stopped by loading all the transport mixture onto a discontinuous gradient of Bromododecane and perchloric acid/sucrose and then centrifuged at 14, 000 ×g for 90 seconds. The discontinuous gradient was prepared by overlaying 1-Bromododecane (800 μL; Sigma-Aldrich) above 100 μL of 20% perchloric acid (Sigma-Aldrich)/8% sucrose solution in 1.5-ml microfuge tube. The samples were snap-frozen in an ethanol-dry ice bath. The bottom part of microfuge tubes containing T cell lysate in perchloric acidsucrose was cut by a microfuge cutter, washed with 300 μL of 0.5%SDS-1%TritonX100, and transferred into scintillation vials. 10 mL scintillation cocktail was then added to each vial, and the radioactivity was then quantitated by liquid scintillation spectrometry.
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