How to extract PBMC cells? Take a look at this sharing

          Peripheral Blood Mononuclear Cell (PBMC) is a type of cell with a single nucleus that exists in the peripheral blood of the human body. They play a crucial role in the immune system, mainly including lymphocytes, monocytes, dendritic cells (DC), and a small number of other cells.
               

1、 Preparation materials:

Before conducting PBMC extraction, the following materials need to be prepared:

1. Human whole blood;

2.15ml centrifuge tube;

3. DPBS;

4. PBMC cell isolation solution (Qida Biotech, catlog: SD0060);

5. PBMC serum-free cell culture medium (Qida Biotech, Catlog: P1901).

2、 Steps for PBMC cell extraction:

1. Transfer 10ml of whole blood into a 50ml centrifuge tube, add 10ml of PBS solution for dilution, and gently mix well;

2. Take two 15ml centrifuge tubes and first add 5ml of lymphocyte separation solution. Then gently add the diluted blood to the upper layer of the standing plate cell separation solution in two centrifuge tubes, being gentle to avoid mixing the two solutions together. Dilute 10ml of blood in each centrifuge tube;

3. 2000rpm, 20min, note that the deceleration setting must be set to no break, or only 10-20% braking. After centrifugation, layers will be obtained as shown in the figure;

4. The cell layer where PBMC is located is white. At this point, the cells in that layer can be aspirated into another clean 15ml centrifuge tube using a straw.

5. Add PBS to 10-15ml, centrifuge at 1500rpm for 10 minutes, remove the supernatant, and then add culture medium for the same cleaning process;

6. Add 5-10ml of culture medium to resuspend cells, and perform subsequent counting culture or plate laying.

If the experiment requires a small amount of cells, the excess cells can be frozen and stored according to the steps of primary cell cryopreservation.

3、 Precautions for separating PBMC cells:

1. Approximately 1 milliliter of peripheral blood can be obtained × 106 mononuclear cells;

2. Ficoll should be in moderation, and peripheral blood should be fully diluted. Temperature directly affects the specific gravity and separation effect of Ficoll;

3. When adding diluted peripheral blood to Ficoll, it should be added slowly to avoid dispersing the interface;

4. When aspirating single-cell layers, excessive supernatant or stratified fluid should be avoided, leading to platelet contamination.

When isolating PBMC cells, we need to pay attention to the above four aspects: estimation of cell quantity, ratio of Ficoll and peripheral blood, temperature control, and operational skills when aspirating cells. Only by achieving these four points can we ensure the smooth progress of the experiment and obtain accurate experimental results.