低速离心连续密度梯度离心Procedure.PPT

Peripheral blood mononuclear cell isolation and preservation PBMC PBMC is a blood cell having a round nucleus, such as a lymphocyte or a monocyte. These blood cells are a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population consists of T cells (CD4 and CD8 postive ~75%), B cells and NK cells (~25% combined). Isolation of PBMC Platelet - 1.030～1.035 Lymphocyte, monocyte - 1.075～1.090 RBC, PMN - 1.090 密度離心： 利用密度介於1.075～1.092的等張溶液 (分層液) 做離心，使細胞按密度分布不同而分離。 常用的分層液：ficoll和percoll兩種(1.077) Ficoll-Hypaque Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll-Hypaque = urografin + ficoll solution (340g/L；density 1.200) (60g/L；density 1.020) Mouse - 1.088；Horse - 1.090 單次密度離心法 Percoll Percoll consists of colloidal silica particles which have been coated with polyvinylpyrrolidone (PVP). Percoll solution (1.135) + phosphate buffer (1:1 mix) 密度 低 高 percoll 高速離心 blood 低速離心 連續密度梯度離心 Procedure 5ml venous blood (EDTA or heparin) + RPMI (1:1稀釋) ↓ 稀釋液緩慢加入含有4ml lymphoprep 的上層中 (血液不可溶入 lymphoprep 中) ↓ RT 離心2500rpm，30 mins (離心機不煞車) ↓ 將上清液吸掉，將PBMC移到另一離心管(盡量不要吸到上下層) ↓ 加入10 ml RPMI wash cell 後離心1500rpm，10mins (x 2) ↓ 加入10ml含10% FBS 的 RPMI 1640 ↓ 將細胞打散，取10ul+ trypan blue（1:1染死細胞） ↓ 利用counting chamber 計數細胞 (cells/ml) ↓ 剩於細胞用細胞離心機離下，並染色 (Liu’s stain) RPMI Roswell Park Memorial Institute medium, commonly referred to as RPMI. A form of medium used in cell culture. It has traditionally been used for growth of Human lymphoid cells. This medium contains a great deal of phosphate and is formulated for use in a 5% carbon dioxide atmosphere. There are a variety of similar media in the RPMI series, such as RPMI 1640. HBSS Hank’s Balanced Salt Solution (HBSS) 平衡鹽溶液(Balanced Salt Solution, BSS) -與細胞生長狀態下的pH值、滲透壓等環境狀態一致。 -具有維持滲透壓、控制酸鹼平衡、供給細胞生存代謝所必需的能量和無機鹽成分等作用。 -使細胞生存並維持一定的代謝基本需要。 Other BSS are