Isolation of PBMCs for gene expression analysis

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of nucleated blood cells, comprising lymphocytes, monocytes, and dendritic cells.

Because these cells are obtained in a less invasive way than biopsies, they have been used in the search for biomarkers in numerous pathologies. Specifically, the gene expression profile of PBMCs has been shown to be a reflection of the physiological and pathological events that occur in different tissues of our body, the expression of certain genes in PBMCs having been related to the development of pathologies such as breast cancer. prostate or diabetes.

Lymphoprep™ Density Gradient

To analyze the gene expression of PBMCs, the first step is to isolate these cells from the whole blood sample taken from the patient. The density gradient with Lymphoprep™ is one of the most used methods for this step.

For this method, 15mL of whole blood is diluted with 15mL of buffered saline supplemented with fetal calf serum. The 30mL of diluted blood is carefully dispensed into a 50mL tube containing 15mL of Lymphoprep™. Then, the 50mL tube is centrifuged at 800 G for 20 minutes at room temperature (15-25ºC). 

Our Unicen 21 and Digicen 21 universal centrifuges by concept, with maximum capacities of 4 x 100 ml, are perfectly adapted to the requirements of this centrifugation step.

As a result of the centrifugation, the cells are divided into 4 layers based on their density, which from bottom to top are: layer with erythrocytes, layer with Lymphoprep™, layer with PBMCs, and layer with plasma. Thus, after centrifugation, the serum layer is discarded to gain access to the PBMCs layer, which is the one of interest. The layer of PBMCs is harvested, and the cells are stored for later use.

Finally, from these isolated PBMCs, cell lysis and RNA isolation would be carried out, and the gene expression profile of said cells would be analyzed, with the technique considered most appropriate in each case.

Density gradient centrifugation is one of the most widely used methods to isolate PBMCs from a whole blood sample, both due to the convenience of the technique, since it consists of few and simple steps, and to the accessibility when it comes to taking the blood sample.