I'm trying to do a project using western blot to assay the level of a cell surface protein that is currently without established norm in pediatric or adult populations. In order to do the assay, I need PBMC from whole blood drawn from pediatric subjects. Does anyone know what the minimum of blood required for optimal PBMC isolation in pediatric population?
Hi Deyin,
For pediatric population, you are limited by IRB restrictions. In most cases in USA and many other countries, IRB allows to draw only 4 ml of blood from pediatric subjecrts. I have done extensive studies with this small amount of blood.
Now you can get 1-2 million PBMC per ml of blood (sometimes little bit more). That means you can get around 10 million cells form 4 ml of blood.
If you don't have to isolate any specific type cell, you have plenty of cells to do western blot. If you have to isolate specific population, that is different story. In, PBMC, there are 3-8% B cells, 20-50% T cells, etc. (more information is available about different subsets of blood cells at (https://www.miltenyibiotec.com/~/media/Files/Navigation/Cell%20analysis/App_note_7Color_Immunophenotyping.ashx)
If you are working with cell surface molecule, I would suggest you to establish a method using FACS. This will allow to interrogate expression of such molecule in different subsets of cells, and you can do these experiment with smaller number of cell (as low as 50-100 thousand cells).
If you have to absolutely depend on western blot, I would suggest to calculate the amount of cells base on sensitivity of you western blot vs proportion of cells you are dealing with.
Hope this helps.
Mrinmoy
Hi Deyin,
For pediatric population, you are limited by IRB restrictions. In most cases in USA and many other countries, IRB allows to draw only 4 ml of blood from pediatric subjecrts. I have done extensive studies with this small amount of blood.
Now you can get 1-2 million PBMC per ml of blood (sometimes little bit more). That means you can get around 10 million cells form 4 ml of blood.
If you don't have to isolate any specific type cell, you have plenty of cells to do western blot. If you have to isolate specific population, that is different story. In, PBMC, there are 3-8% B cells, 20-50% T cells, etc. (more information is available about different subsets of blood cells at (https://www.miltenyibiotec.com/~/media/Files/Navigation/Cell%20analysis/App_note_7Color_Immunophenotyping.ashx)
If you are working with cell surface molecule, I would suggest you to establish a method using FACS. This will allow to interrogate expression of such molecule in different subsets of cells, and you can do these experiment with smaller number of cell (as low as 50-100 thousand cells).
If you have to absolutely depend on western blot, I would suggest to calculate the amount of cells base on sensitivity of you western blot vs proportion of cells you are dealing with.
Hope this helps.
Mrinmoy
Mrinmoy, thank you so much for your answer and help! That was very informative. I will definitely look into FACS. Thank you again!
According to a number of paediatric, REB approved clinical trials here in Canada > or = to 5mL of whole blood should be sufficient depending on their immune status. This will yield ~6-11x10^6 PBMCs. If you are new to this, I suggest investing in SepMate tubes vs. just 50mL Falcons.
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