Cells separated from peripheral blood, bone marrow, peripheral lymphoid organs or various tissues are a mixture of multiple cells. If you want to study the structure, function or surface markers of a specific cell, you must first separate and purify the cells. Cell separation can be performed by density gradient sedimentation, attachment, affinity chromatography, ring formation, complement-mediated killing and lysis, immunomagnetic beads and flow cytometry (FCM) according to the differences in size, density, surface charge, adsorption capacity or surface antigens.

Ⅰ. Principle of PBMC Separation by Ficoll-Hypaque Density Gradient Centrifugation

The density of the layering fluid commonly used to separate human peripheral blood mononuclear cells (PBMC) is 1.077 ± 0.001 kg/L of polysucrose (Ficoll)-Urografn (F/H) layering fluid. Ficoll is a polymer of sucrose, which is neutral, highly hydrophilic, and has an average molecular weight of 400,000. When the density is 1.2 g/ml, it still does not exceed the normal physiological osmotic pressure and does not penetrate the biological membrane. Red blood cells and granulocytes have a high specific gravity and sink to the bottom of the tube after centrifugation; the specific gravity of lymphocytes and monocytes is less than the specific gravity of the layering fluid. After centrifugation, they float on the surface of the layering fluid. A small number of cells may also be suspended in the layering fluid. By absorbing the cells on the surface of the layering fluid, mononuclear cells can be separated from peripheral blood.

II. Experimental Steps for PBMC Separation

1. Add an appropriate amount of lymphocyte separation solution to the short medium tube.

2. Take heparin anticoagulated venous blood and mix it thoroughly with an equal amount of Hanks solution or RPMI1640 solution, and slowly superimpose it on the layered liquid surface along the tube wall with a dropper, paying attention to maintaining a clear interface. Horizontal centrifugation at 2 000 rpm, 20 min.

3. After centrifugation, the tube is divided into three layers, the upper layer is plasma and Hanks solution, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation solution. At the interface between the upper and middle layers, there is a narrow band of white cloud layer dominated by mononuclear cells, which include lymphocytes and monocytes. In addition, it also contains platelets.

4. Use an elbow dropper to insert into the cloud layer and carefully absorb the mononuclear cells; you can also first discard the upper layer of RPMI 1640 and plasma, and then collect the cloud layer rich in PBMC. Place the cloud layer cells in another short medium tube, add more than 5 times the volume of Hank's solution or RPMI 1640, centrifuge at 1500 rpm for 10 minutes, and wash the cells twice.

5. After the last centrifugation, discard the supernatant, add RPMI 1640 containing 10% calf serum, and resuspend the cells. Take a drop of cell suspension and mix it with a drop of 0.2% trypan blue dye, and count the total number of cells in four large squares on the hemocytometer. Mononuclear cell concentration (cell number/1ml cell suspension) = total number of cells in 4 large squares/4×10^4×2 (dilution multiple).

6. Cell viability test: dead cells can be stained blue, and live cells are not stained. Count 200 lymphocytes. Calculate the percentage of live cells. Percentage of live cells (%) = number of live cells/total number of cells×100%.

7. Cell culture: After counting the cells, adjust the cell concentration to 2×10^5/ml culture medium and add it to a six-well plate or a 24-well plate for culture.

8. Cell collection: Aspirate the culture medium in the six-well plate or the 24-well plate and discard it, add 200μL Trizol to each well, blow the well wall several times with a pipette, and transfer the Trizol into the EP tube.

9. Cell freezing: Store the collected cells in a -80℃ refrigerator.

Ⅲ. Precautions During the Experiment

1. Ficoll should be used in an appropriate amount and peripheral blood should be fully diluted.

2. Temperature directly affects the specific gravity and separation effect of Ficoll.

3. When adding diluted peripheral blood to Ficoll, add it slowly to avoid breaking up the interface.

4. When aspirating the mononuclear cell layer, avoid aspirating too much supernatant or layering liquid to cause platelet contamination.

IV. Advantages of Tek Biotech's Alpaca PBMC Products

1. High cell viability: The separation reagents used in the experiment are sterile, low in endotoxin, and do not cause damage or stimulation to the cells. The operators have many years of experience in PBMC cell separation, which can ensure the original state of the cells to the greatest extent. The viability of fresh cells after separation or cryopreservation and resuscitation can reach more than 90%.

2. High product safety and no infectious source: Strictly control the source of alpaca/camel whole blood to ensure that the experimental camels have undergone relevant infectious source testing, providing customers with safer and more reliable experimental materials.

3. Clear and traceable cell sources: The whole blood used to separate alpacas/camels is collected by regular animal centers, with a fully compliant procurement process and whole blood biosafety certificate, eliminating the worries of customers.

Tek Biotech has mature animal immune technology and PBMC separation technology to ensure that cells are not damaged or stimulated during separation. The subsequent purification technology ensures the purity and viability of camel mononuclear cells and other technical indicators, which are suitable for subsequent cell culture, antibody preparation and other research.