Percoll-based Preparation Of Synaptosomes From Rat Spinal Cord

Objective : To construct a rapid,stable and efficient method preparing of rat spinal synaptosomes based on Percoll gradient procedure.Methods: Extract the required spinal cord without cauda equina from the killed animal.Rinse several times in excess volumes of ice-cold surcose/EDTA buffer to reduce blood levels.Remove as much liquid as possible using filter paper.Add 9 ml of ice-cold surcose/EDTA buffer to 1 g of fresh spinal cord tissue in a small glass beaker.Pour the tissue into a 15-ml glass tissue grinder and homogenize with ten even strokes.Centrifuge the homogenate at 1000 g for 10 min at 4? in a 10-ml polycarbonate centrifuge tube.Dilute the S1 fraction obtained to 10–14 ml with surcose/EDTA buffer and keep on ice and measure the protein content using BCA to 4-5 mg/ml.Slowly and carefully pipette 2 ml of the diluted S1 fraction over the top 3% layer in each tube containing the Percoll gradients.Centrifuge the tubes at 19500 g for 10 min(the deceleration of brake should be set to 500 r.p.m at least).Collect the high density area(F3,F4)between the layers of 10% Percoll,15% Percoll,and 23%Percoll,then dilute F3,F4 with PBS buffer in a beaker and centrifuge at 14000 g for 10 min.Discard the supernatant and the pellet was resuspended in PBS for use.The prepared suspension was prepared into standard electron microscopy which was observed by electron microscopy.Western blot(WB)was used to analyze the expression of synaptophysin and PSD95.Results: The fractions are easy to see as cream-colored membranes that sit at the interface between each Percoll layer.The morphological integrity of the synaptosome of the spinal cord was observed under the electron microscope.Synaptosomes were very densely packed with synaptic vesicles and contained well-preserved intrasynaptosomal mitochondria.Presynaptic components,synaptic clefts,and postsynaptic densities remain intact.Through the Invitrogen E-Gel Imager,the gray scale of Synaptophysin,PSD95 and ?-actin were clearly observed.Conclusion: In this research,preparing synaptosome of spinal cord based on discontinuous Percoll density gradient centrifugation is time-saving,convenient,and efficient.