Why does Ficoll density gradient centrifugation fail to separate PBMCS?

Adding blood to the lymphocyte isolation solution is crucial. The entire liquid level must be kept flat. The nozzle of the centrifuge tube is tilted at a 45 degree Angle, and then at a very slow rate, drop by drop against the wall of the centrifuge tube, slowly adding a few drops to form an interface. The whole process requires a very steady hand.

1 Initial PBMC estimates that 1 milliliter can be divided into 1 million cells. Conventional 5 ml peripheral blood can be divided into 5*10^6-1*10^7 cells, and the peripheral blood volume can be determined according to your own needs.

2. The ratio of the separated solution to the diluted blood was 1:2. After peripheral blood is obtained, density gradient centrifugation is performed, and it is best to dilute it in a ratio of 1:1. In this way, the separation effect can be greatly improved from 700g to 1000g. The separation rate of 800 to 900g is the highest (g*2.5= RPM, that is, 2000-2500rpm).

Since the specific gravity of each component in blood is different, when Ficoll-Hypaque mixture with a specific gravity of 1.077 close to isotonic is used as a density gradient centrifugation, the various blood components will reaggregate according to the density gradient. Plasma and platelets are suspended in the upper part of the stratified fluid because of their low density. Red blood cells and granulocytes sank to the bottom of the stratified fluid because of their greater density. The density of the PBMC is slightly lower than that of the stratified liquid, so it is located at the interface of the stratified liquid, so that the PBMC can be obtained. Generally, it is centrifuged with a horizontal centrifuge at 2500rpm/min(depending on the model) for 20min. This centrifugation is critical.