I would like to isolate PBMCs including neutrophils from the whole blood? Your suggestions wills be appreciatable..Thanks
This has been asked on several threads. I've include only a few.
https://www.researchgate.net/post/How_do_you_best_separate_white_blood_cells_to_get_all_the_cell_fractions_for_further_experiments
https://www.researchgate.net/post/Can_anyone_help_with_human_neutrophil_extraction
https://www.researchgate.net/post/Does_anyone_know_a_protocol_for_primary_human_neutrophil_isolation
Dear you can isolate white cells by centrifugation and collecting the buffy coat.
you can use Ficoll Paque density gradient centrifugation and isolate the buffy coat. You can use the cells fresh or freeze them at density not greater than 2 * 10^8/ml.
Shen-An Hwang :Many thanks for the link and reply. I have gone through the thread which you have sent me the link. There is no answer for my concern. I need to isolate together of lymphocytes, monocytes and neutrophils. So which method is appropriate to isloate all three cell types?
Emmanuel Ifeanyi Obeagu : Thanks for the reply. Does the buffy coat contains neutrophils which you were mentioned ?
Kawther K. Ahmed : Thanks for the reply. Does the buffy coat contains neutrophils which you were mentioned ? I need to isolate together of lymphocytes, monocytes and neutrophils. So which method is appropriate to isloate all three cell types?
You can use the PBMC protocol, and the layer will contain monocytes and lymphocytes. The granulocyte will be with the RBCs.
If you don't want to lyse the RBCs, which I understand is a bit of an issue, you can use a double gradient. People have done it with Ficoll, but I don't remember the densities. What I used to use is Histopaque 1077 layered on top of Histopaque 1119. The cell layer between the plasma and 1077 is the monocytes/lymphocytes. The cell layer between 1077 and 1119 are the granulocytes.
https://pdfs.semanticscholar.org/8d74/07a1dbdd3d11a80a387e02d7de15e93c5b01.pdf
The link should help.
There are at least three commercial variants available that will leave all types of white blood cells (including granulocytes) but remove erythrocytes.
One is based on a Ficoll like substance but with a slightly different density so that neutrophils will be layered with other white blood cells while the erythrocytes precipitates (one manufacturer is pluriBead, but I think other manufacturer may have similar products https://www.pluriselect.com/se/leuko-spin-medium.html).
The other is based on precipitation of erythrocyte through binding to Hetastarch or similar substances and will precipitate erythrocytes but leave white blood cells in the supernatant (for example HetaSep from Stem cell technologies https://www.stemcell.com/products/hetasep.html).
The third is more usable for smaller volumes as it is a bit more expensive, but will remove erythrocytes magnetically based on antibody-labeled magnetic particles. (for example EasySep RBC depletion reagent: https://www.stemcell.com/easysep-rbc-depletion-reagent.html).
Using the Ficoll gradient density method, layer whole blood on ficoll and centrifuge. Buffy coat contains PCMCs (lymphocytes and monocytes) and just on top the red cell layer are neutrophils. Lyse the red cell layer to yield neutrophils.
Dear Pradeep,
The commercial kits are very nice but if you want a more cost-effective solution you can also just treat the whole blood with ice-cold ACK lysis buffer (homemade or commercial) to lyse the RBCs. Then you're left with all WBC types. No need to first do a density gradient. As long as you incubate with the lysis buffer on ice and don't incubate for extended times (I would typically incubate for 20min), your WBC will be fine. Of course there is some loss but you will have some loss with any method.
Good luck!
Erinke
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