Self forming iodixanol density gradients of human red blood cells
The subcellular distribution of annexin A7 was addressed according to [24]. Red blood cells were centrifuged and washed in isotonic buffered salt solution, collected in ice cold lysis buffer (20 mM Hepes, pH 7.4, 200 μM CaCl2, 0.25 M sucrose, and proteinase inhibitors (Roche)), homogenized by a loosely fitting dounce homogenizer and additional passages through a 22 gauge needle. A 50 % iodixanol solution was established by mixing Optiprep solution (60 % iodixanol; Sigma) with buffer (120 mM Hepes, pH 7.4, 1.2 mM CaCl2, 0.25 M sucrose). Seven parts of this 50 % iodixanol solution were mixed with thirteen parts of the red blood cell solution, generating a gradient solution containing 17.5 % iodixanol. The mixture was filled into a centrifuge tube underlayed by cushions of 30 % and 35 % iodixanol and centrifuged for 3 hours at 270,000 × g in a SW41 Ti swing out rotor (Beckman) to achieve a nearly linear density gradient (approximately 1.06 g/ml to 1.20 g/ml) [24]. Fractions of 0.6 ml were collected from the top of the gradient by a 1 ml syringe without using a needle and immediately frozen in liquid nitrogen for further use.