How is a mouse spleen cell preparation carried out?
For the preparation of mouse spleen cells by mechanical disruption, it is recommended to cut the spleen once and to pass the tissue through a steel mesh by using a plunger. Subsequently, the cells are flushed into a petri dish containing PBS. Collagenase treatment is strongly recommended for the isolation of dendritic cells and liver sinusoidal endothelial cells to increase recovery of the cells. In order to remove fat, cell debris and cell aggregates, the cells can be passed through a cotton wool column. With this procedure, each spleen should yield approximately 5×107–1×108 cells. Due to relatively low numbers of erythrocytes in the spleen cell preparation (2:1 ratio for erythrocytes:leukocytes, compared to a 1000:1 ratio in human peripheral blood), an erythrocyte lysis is not required.
How can dead cells be removed from a mouse spleen cell preparation?
Dead cells can be removed from mouse spleen cell preparations prior to magnetic labeling by gradient centrifugation. Dead cells will be separated at a density of 1.36. Alternatively, dead cells can be removed using the Dead Cell Removal Kit.
Should EDTA be added to the buffers for mouse cell preparation?
For separation of mouse cells with MACS® Technology, the addition of 2 mM EDTA to the MACS Separation Buffer (PBS, 0.5% BSA, pH 7.2) is recommended.
Is a special sample preparation protocol required for mouse cells?
Generally, a special sample preparation protocol for the isolation of mouse cells with MACS® Technology is not required. Exceptions are the isolation of dendritic cells and liver sinusoidal endothelial cells (LSECs) where collagenase treatment is strongly recommended to increase the recovery of the cells. For the isolation of LSECs from liver, a density gradient centrifugation has to be performed. A single-cell suspension is the prerequisite for optimal separation results. Cell aggregates may decrease the purity of the positive fraction as they may contain both target cells and non-desired cells. In order to remove cell aggregates, the cells should be passed over a Pre-Separation Filter before loading them onto a cell separation column.