In order to ensure the yield and purity of PBMC while separating quickly, several aspects need to be paid attention to:

一．Selection of Anticoagulants:

If ELISPOT experiments and cell culture experiments are required later, heparin anticoagulants need to be selected when collecting blood, because EDTA anticoagulants inhibit coagulation by chelating calcium, and chelated calcium may impair cytokine secretion during antigen-specific restimulation.

二．Optimal Separation Temperature

The optimal separation temperature of the separation product, including the separation tube, is (20±5)℃. When the temperature is too high, the density of the separation solution mainly composed of sucrose will decrease, and some lymphocytes will enter the separation layer; if the temperature is too low, the density of the separation solution mainly composed of sucrose will increase, and red blood cells will also reduce aggregation at low temperatures. Therefore, compared with room temperature conditions, red blood cells are difficult to precipitate below the separation solution layer and are easily mixed into the PBMC layer. Therefore, during the separation process, it is necessary to ensure that both the blood sample and the separation tube are at room temperature.

三．Precautions for Refrigerated Blood Separation

If frozen blood is used for separation, there will usually be more red blood cells in the PBMC layer, and the activity of PBMC will be affected to a certain extent. It is recommended to restore the blood sample to room temperature before separation and extend the centrifugation time appropriately, for example, 30 minutes. It should be noted that if the blood sample is refrigerated for a long time, even if the blood sample has been restored to room temperature, the centrifugation time has been extended, and the separation effect cannot be compared with fresh blood samples. It is recommended to store blood at room temperature for no more than 2 hours.

四．Amount of Blood Sample

Red blood cells are used to replace the separation solution originally located at the bottom of the tube to the top of the red blood cells. The separation solution pushes the PBMC and plasma with relatively small density to the top of the sieve plate. The number of red blood cells in the blood is fixed. If the amount of blood is too small, the replacement effect of red blood cells after separation is poor, and PBMC may be close to the sieve plate in the separation tube and difficult to aspirate. At this time, even diluting the blood sample cannot improve the separation effect. Therefore, it is not recommended to use red blood cell sedimentation fluid with separation tubes. After sedimentation treatment, the amount of red blood cells decreases and the replacement effect is poor.

If the amount of blood is too much, it may cause the red blood cells to exceed the sieve plate, the PBMC layer is blurred, and the separation effect is reduced. In summary, the recommended amount of blood sample for a 15mL separation tube is 3mL~6mL; the recommended amount of blood sample for a 50mL separation tube is 15mL~25mL.

五．Methods for Removing PBMC

After using a separation tube to separate PBMC, you can choose to pour out or aspirate PBMC directly. If you need a relatively higher cell yield, you can choose to directly pour out the liquid above the sieve plate; if you need a relatively higher cell purity, you can choose to aspirate PBMC with a Pasteur pipette or a pipette.

六．What Method is used to Count PBMC

AO/PI: nucleated live cells and nucleated dead cells can be counted separately, and red blood cells are not stained. AO can pass through the intact cell membrane and embed into the nuclei of all cells (live and dead cells), showing green fluorescence; PI can only pass through the incomplete cell membrane, that is, the cell membrane of dead cells, and embed into the nuclei of all dead cells, showing red fluorescence. At this time, live/dead PBMC can show fluorescent signals. Mature red blood cells and platelets cannot be stained by AO/PI because they do not have nuclei, so they can be completely excluded and not counted.

Methylene blue acetate: 3% methylene blue acetate can count nucleated cells, but cannot distinguish between live and dead cells. 3% acetic acid can destroy cell membranes; methylene blue can bind to nucleic acids and stain cell nuclei. During the cell counting process, the cell membranes of all cells will be lysed, but red blood cells do not have nuclei, so they will not be counted. Although the 3% methylene blue acetate counting method cannot distinguish between live and dead cells, generally speaking, there are more red blood cells than dead cells, so this counting method can also be used.

Trypan blue: The cell membrane structure of normal living cells is intact, trypan blue cannot enter the cell, and the living cells are transparent; usually when the cell membrane integrity is lost, it can be considered that the cell has died. Therefore, for dead cells or cells with incomplete cell membranes, the cell membrane permeability increases, and trypan blue can enter the cell and dye the cell blue. Therefore, trypan blue can distinguish between live and dead cells, but cannot distinguish between red blood cells and nucleated cells.

TekBiotech has mature animal PBMC separation technology to ensure that cells are not damaged or stimulated during the separation process. 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.

PBMC Separation

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 purified by density gradient sedimentation, attachment, affinity chromatography, ring formation, complement-mediated killing and lysis, immunomagnetic beads and flow cytometry (FCM) according to differences in size, density, surface charge, adsorption capacity or surface antigens.

一．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.

二．Experimental Steps for PBMC Separation

1. Add an appropriate amount of lymphocyte separation fluid 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. Use a dropper to slowly superimpose it on the layered liquid surface along the tube wall, and pay attention to maintaining a clear interface. Horizontal centrifugation at 2,000 rpm for 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 fluid. At the interface between the upper and middle layers, there is a narrow band of white cloud layer dominated by mononuclear cells. Mononuclear cells 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.

三．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 dispersing the interface.

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

四．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 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.