How to Isolate PBMCs from Whole Blood Using Density Gradient Centrifugation (Ficoll™ or Lymphoprep™)
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- čas přidán 29. 08. 2024
- This step-by-step technical guide demonstrates how to isolate peripheral blood mononuclear cells (PBMCs) from whole blood using density gradient centrifugation (e.g. Ficoll-Paque™ or Lymphoprep™).
In this video, you can see how to layer blood over the density medium and learn two different ways of harvesting cells from the blood plasma: density medium interface.
View a written protocol on how to isolate mononuclear cells from whole blood by density gradient centrifugation: bit.ly/3k8bZZC
Learn more about Lymphoprep™ density gradient medium: bit.ly/3mVMxs2
Learn how SepMate™ tubes can speed up and simplify your density gradient centrifugation at www.SepMate.com.
After isolating PBMCs, you can isolate highly purified cell subsets using the EasySep™ magnetic cell separation. Learn more at www.EasySep.com.
#PBMCisolation #CellSeparation #CellIsolation
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Thanks. Obrigado.
Interesante video. Muy didactico y reproducible para el pregrado.
If I just use whole blood that hasn't been diluted with PBS, will the protocol be the same?
If you does not wish to dilute the blood, you do not need to do so, although recovery might be compromised. This is because when erythrocytes in whole blood are aggregated, some lymphocytes are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap lymphocytes is reduced by diluting the blood. Dilution gives a better lymphocyte yield and reduces the size of the cell clumps. Additionally, if you are using SepMate™ tubes, not diluting the blood may result in poor red blood cell depletion and tube overload.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
Hi! Is there any reason why the whole blood preparation and the Ficoll shouldn't be mixed before centrifugation? I don't quite undestand this phase because in the centrifugation, they will be mixed anyway. Thanks!
Ficoll™ and other density gradient media act as a layer to separate cell types based on their density. During centrifugation, cells more dense than Ficoll™ (such as red blood cells and granulocytes) migrate below the Ficoll™ layer and pellet at the bottom of the tube. Lymphocytes, monocytes and platelets are not dense enough to penetrate the Ficoll™ layer. These cells therefore collect as a concentrated band at the interface between the original blood sample and the Ficoll™. If the blood sample is mixed with the density gradient medium before centrifugation, this density-based separation cannot happen. If you have any more questions, feel free to email us at techsupport@stemcell.com.
Thank you, a good explanation
Thank you very much for this very nice video. I am currently following the same process but with sheep blood. The point is I don't have 4 layers after the first centrifuge. Does anybody knows if the type of blood plays an important part in this experience ? thank you to reply me
Its been three years
Is all going good now ???
Good
I Purchased PBS from stemcell tech, what do i need to add to make it 2% FBS.
What will be the problem if we use only PBS w/ no 2%fbs?
For the dilution buffer to be used with density gradient separation, the 2% FBS is typically included to support the cells during the separation process. You may leave it out, but there may be reduced viability for the cells. The buffer is 2% FBS by volume. For 100ml of dilution buffer, you would use 98ml of PBS + 2ml FBS. You may then sterile filter, or not, as your lab requires. If you have any more questions, feel free to email us at techsupport@stemcell.com!
Can we use ficoll insted of histopaque solution?
Yes, you can use Ficoll™, Lymphoprep™, Histopaque®-1077 or any other density gradient medium intended for isolating mononuclear cells with a density of 1.077g/mL. If you have any more questions, feel free to email us at techsupport@stemcell.com.
How is a lymphocyte isolated from whole blood?
Hi, lymphocytes can be found at the interface of the density medium and plasma after centrifugation. This layer can be collected using a pipette as shown in the video. If you have any further questions about the procedure or products, feel free to email us at techsupport@stemcell.com.
How much ficoll is to add for 5 ml blood with 5 ml PBS.
Hi Pradeep, for recommended volume and tube sizes to use with Lymphoprep™, please refer to Table 1. Recommended Volumes and Tube Sizes in the Product Information Sheet for Lymphoprep™ (cdn.stemcell.com/media/files/pis/10000000358-PIS_05.pdf?_ga=2.212220372.1312914144.1666602988-1504112607.1666216542&_gac=1.256261753.1666346489.Cj0KCQjwhsmaBhCvARIsAIbEbH4yFJrn2h4F6TQBDpLhmF5EEA93zzpmtNXkIcLWdcWN8eF2ljsgcjoaAhotEALw_wcB). For 5mL of whole blood combined with 5mL of PBS, 10mL of Ficoll™ or Lymphoprep™ should be used with a 50mL tube. If you have any further questions, feel free to email us at techsupport@stemcell.com.