I am preparing Ficoll solution. I weighed 1 g of Ficoll powder and put it into a beaker. Then I added 5 ml of PBS and mixed. After completely dissolving the Ficoll, I added another 5 ml of PBS, thus preparing 10 ml of Ficoll solution, and kept it aside.
After that, I collected blood. I took a 15 ml test tube and added 3 ml of blood and 3 ml of PBS to dilute it. Then, in a separate test tube, I took the Ficoll solution and carefully layered the diluted blood slowly on top of the Ficoll solution. But instead of staying as separate layers, the blood and Ficoll got mixed together.
I then centrifuged it at 1500 rpm for 30 minutes, but the PBMC layer did not form.
Dear Vennela Marla,
I suggest using the Ficoll/Pancoll/Biocoll solution to minimize variability.
What I usually do is the following: after adding 15 mL of Biocoll solution to a 50 mL tube (either a regular Falcon or a SepMate™ PBMC Isolation Tube), I carefully add 15 mL of blood diluted 1:1 in PBS, making sure not to disturb the Biocoll layer. I then centrifuge for 30–35 minutes at 440 × g (with the brake off for regular 50 mL tubes, or set to 2 for SepMate tubes). After centrifugation, the PBMC layer should be visible.
I hope this works for you, and I am happy to answer any further questions.
Dear Vennela Marla
The Ficoll solution has a specific density that allows for the separation of blood components during centrifugation. You may be using a Ficoll solution with the wrong density that may be interfering with the separation process.
The main challenge is achieving the precise density needed to separate lymphocytes. Preparing a custom Ficoll solution requires careful weighing, dissolving, and density adjustment, which can be complex and prone to error. If the density is too high, cells might not be able to penetrate the gradient as expected, leading to a low yield of desired cells. On the other hand, if the density is too low, cells, particularly lymphocytes, can penetrate too deeply, leading to mixing. Commercial solutions already contain the necessary components (Ficoll polysaccharide and sodium diatrizoate) to achieve the optimal density and osmolarity for separating PBMCs.
To prepare a Ficoll solution for lymphocyte separation, it is best to use a commercially prepared density gradient medium like Ficoll-Paque or HISTOPAQUE, which are pre-adjusted to a specific density (e.g., 1.077 g/mL). You then layer diluted whole blood over this medium in a centrifuge tube and spin at room temperature with the brake off, causing lymphocytes to collect at the interface between the plasma and the gradient, allowing for their isolation.
Best,
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