Dissociation of Cells from Primary Tissue

实验概要 Obtain single cell suspensions from primary tissue.

主要 试剂 0.25% trypsin soybean trypsin inhibitor  

主要设备 stainless steel mesh [Details：100 to 200 µM ]  

实验步骤 1.After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. Allow the tissue pieces to settle, and remove the supernate. Repeat the wash 2 or 3 times. 2.Place the container with the tissue pieces on ice, and remove any remaining supernate. Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (1 ml of trypsin for every 100 mg of tissue). 3.Incubate at 4°C for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity. 4.Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min. 5.Add warm, complete media to the tissue pieces and gently disperse the tissue by pipetting. If using a serum-free medium, also add soybean trypsin inhibitor. 6.Filter the cell suspension through sterile, stainless steel mesh (100 to 200 µM) to completely disperse any remaining tissue. Count and seed the cells for culture.