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sir iam going to do pbmc isolation for the firsttime using histopaque...
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Lekshmipriya P Aug 5, 2021
sir iam going to do pbmc isolation for the firsttime using histopaque method . i want to know the quantity of RPMI media required resuspend the pellet after washing and what all supplements have to added into the RPMI media
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Santosh K Panda Author Answered Aug 10, 2021
Infectious Disease Biology, Institute of Life Sciences, India
Hi Lekshmipriya,
The amount of media required to resuspend varies based on your requirement of cell concentration. To start, resuspend with 500 ul of media if you are isolating from 5 ml of blood. 1ml if isolating from 10 ml of blood. Count the cells and then adjust.
Here is the recipe of RPMI (usual complete RPMI)
Filter sterilize the following:
500 ml RPMI 1640 Medium Sigma
50 ml FBS
5 ml 1M HEPES Buffer
5 ml Kanamycin (100x)
5 ml Glutamax (100x)
5 ml sodium pyruvate (100x)
5 ml Nonessential AAs (100x)
The amount of media required to resuspend varies based on your requirement of cell concentration. To start, resuspend with 500 ul of media if you are isolating from 5 ml of blood. 1ml if isolating from 10 ml of blood. Count the cells and then adjust.
Here is the recipe of RPMI (usual complete RPMI)
Filter sterilize the following:
500 ml RPMI 1640 Medium Sigma
50 ml FBS
5 ml 1M HEPES Buffer
5 ml Kanamycin (100x)
5 ml Glutamax (100x)
5 ml sodium pyruvate (100x)
5 ml Nonessential AAs (100x)
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