Horse PBMCs

As required by institutional and ethical guidelines, the animal is anesthetized before blood collection. Blood collection can be accomplished depending on the size of the animal. For small animals such as rats and mice, blood is typically collected from the tail vein, retro-orbital sinus, or by cardiac puncture. For larger animals such as non-human primates, blood is commonly collected from a vein, mainly the saphenous or femoral vein. To prevent clotting, an anticoagulant is added immediately to the blood. Heparin or EDTA is usually used as anticoagulant. The blood is then gently mixed by inverting the tube several times. Vigorous shaking is avoided to prevent hemolysis.

After addition of the coagulant, the blood is diluted with an equal volume of PBS or sterile saline. This step enables the reduction of cell density to facilitate better separation in the gradient. The solution is then gently mixed by inverting the tube.

A density gradient medium is prepared by adding Ficoll-Paque or Histopaque to a sterile 15 mL or 50 mL centrifuge tube. Approximately 3 mL of density gradient medium per 10 mL of diluted blood is then used. The diluted blood is then layered carefully on top of the density gradient medium. By adding blood along the side of the tube using a pipette, mixing of the mixture is avoided. Next, the mixture is centrifuged at 400 x g for 20-30 minutes at room temperature. Gentle layer separation is ensured by not using the centrifuge brake.

The PBMC layer is identified based on its position alongside three other components. The top layer is composed of plasma. The second layer is thin and white, representing the PBMC layer. The third layer is made up of the density gradient medium which is either Ficoll or Histopaque. Finally, the fourth and bottom layer is composed of red blood cells. The second layer, which is the PBMC layer, is carefully harvested using a pipette and then transferred into a new, clean 15 mL centrifuged tube.

Once the PBMCs have been collected, PBS or culture medium is added to dilute the Ficoll or Histopaque. Next, the solution is centrifuged at 300 x g for 10 minutes at room temperature to pellet the PBMCs. After discarding the supernatant, the cell pellet is gently resuspended in fresh PBS or culture medium. This is followed by two more steps of washing to make sure no Ficoll/Histopaque and other contaminants remain.

To measure viability, cells are first counted using a hemocytometer or an automated cell counter. The cell concentration can be adjusted depending on downstream experiment requirements. Then, cell viability assessment is performed using trypan blue staining or another similar method. Ideally, the viability of PBMCs for functional assays should be above 90%.

If the freshly collected PBMCs are to be used immediately, cells are resuspended in an appropriate medium or buffer for further processing for techniques such as flow cytometry, cell culture, etc. If the PBMCs are to be stored for later use, the cells are frozen in a cryoprotective medium such as 10% DMSO in fetal bovine serum and then gradually cooled to -80°C before transferring to liquid nitrogen for long-term storage. Sources: Fuss et al. (2009), Reidhammer et al. (2016). We cryopreserve the cells in serum-free cryopreservation media to prevent potential effects of growth factors before and during international shipping. Let us know if you have special requests and we will be glad to accommodate them.