Hi all,
I'm trying to isolate PBMC from the blood of several congenic mice (blood samples are mixed). By using ficoll, erythrocytes do never cross the ficoll layer and are just staying at the interface (with 1.086 ficoll) or inside the ficoll layer (with 1.077 ficoll)... But no PBMC layer is visible in any case !
Could the blood mixing be at the origin of the problem ? We do not have an animal facility in house and I have to order fresh blood from a third-party provider, so it's not really possible to get individual tubes containing blood from individual mouse...
And, as the objective is to obtain monocytes for macrophage differentiation, I'm not sure I can use splenocytes for doing such an experiment...
(sorry, my skills are more in Human immunology than in Mouse experimental skills, as you can see... ;-) ).
Thank you !
Hi Alexis,
You need to do an RBC lysis before putting the cells on the ficoll. You can use ACK lysis buffer which you can make yourself. After lysis just layer the remaining cell suspension on top of the ficoll and centrifuge. The neutrophils will be in a pellet at the bottom and the mononuclear cells will be in a layer in the gradient.
Just to warn you, it is not easy to get PBMCs from mouse blood after the ficoll separation in any sort of significant amounts. I tried it many times and did not have very good luck. See the attached paper for actual numbers.
Good Luck!
If yield and recovery are an issue you may want to consider magnetic separation. For mouse derived monocytes you would probably be better off starting with bone marrow.
Thanks Tom for the information and for the paper. I've already seen this paper from 1978, but they use whole blood without lysing the RBC, and seem to get reasonable number of PBMC afterwards...
But you're right, performing a RBC lysis before ficoll can be a way to finally obtain PBMCs in my next try. I'll definitely consider this option.
In this paper from Chi and Harris, they also isolate PBMCs from individual blood, which is maybe the origin of my troubles... But unfortunately, my study design and biological sourcing constraints will not allow me to get blood samples from individualized mice...
Thanks Benjamin for your help. I'm afraid that I won't have the possibility to use mouse bone marrow in my assay design, but I keep this option in mind.
I also had poor success with obtaining good PBMC isolation from mouse blood. One thing I tried was increasing the volume of blood with PBS and then overlaying on Ficoll with a 2:1 ratio. I would collect 100-200µl of blood, increase volume to 1mL with PBS and then overlay on 500µl of Ficoll in a 2mL eppendorf tube.
Thanks for your help, Greg.
I think I'll give up with the idea of mixing blood from several animals before ficolling, and go for more "individual" approaches.
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