Can anyone explain, which source is best for isolation of mouse CD4+ T cells for basic functional assays, spleen or lymph nodes?
In addition, I haven't noticed anyone isolating CD4+ T cells from mouse peripheral blood. Is there a particular reason for this?
Lymph nodes are best if you are looking for naive T cells which are "pre-enriched" as you will get a higher percentage of T cells, but lower yield, from the LN. I typically harvest the brachial,axial, inguinal, cervical from naive mice(~5x106 cells total). Keep in mind that the mesenteric LN's are huge and will produce a lot of cells... but have a more activated status, not as naive in phenotype. It can really pump up your yield if you go for those. To harvest the mesenterics, locate the appendix and place if over the top of the heart. The large and small intestine should now be at 90' angle cascading down over the liver. The color contrast of the liver makes it easier to find the mesenteric LN right in the middle of the integument between the large/small intestine.
If you are just looking for a lot of cells quickly, go for the spleen. ~108 cells, of which you get ~107 CD4+ T cells with a purification kit. DYNAL or Miltenyi were the ones I typically used.
On average, it takes me about 30seconds to grab a spleen and ~5min to harvest all the LN. Here is a link to help with locating the LN's in the mouse.
http://www.informatics.jax.org/greenbook/images/13-4.jpg
For a naive mice the easiest would be the spleen, there you could isolate CD4+ cells in big numbers. Lymphnodes in a naive mice are difficult to find and they usually contain very few cells, same applies for the blood.
If you have an immunized mice, then you can go for LNs and you can for example re-stimulate the isolated cells, or co-culture them with APCs etc. For basic functions you can also pool LN and spleen cells.
Lymph nodes are best if you are looking for naive T cells which are "pre-enriched" as you will get a higher percentage of T cells, but lower yield, from the LN. I typically harvest the brachial,axial, inguinal, cervical from naive mice(~5x106 cells total). Keep in mind that the mesenteric LN's are huge and will produce a lot of cells... but have a more activated status, not as naive in phenotype. It can really pump up your yield if you go for those. To harvest the mesenterics, locate the appendix and place if over the top of the heart. The large and small intestine should now be at 90' angle cascading down over the liver. The color contrast of the liver makes it easier to find the mesenteric LN right in the middle of the integument between the large/small intestine.
If you are just looking for a lot of cells quickly, go for the spleen. ~108 cells, of which you get ~107 CD4+ T cells with a purification kit. DYNAL or Miltenyi were the ones I typically used.
On average, it takes me about 30seconds to grab a spleen and ~5min to harvest all the LN. Here is a link to help with locating the LN's in the mouse.
http://www.informatics.jax.org/greenbook/images/13-4.jpg
Thank you guys, you have been most helpful. Special thanks to David for your comprehensive explanation!
Would you happen to know by any chance how come mouse peripheral blood contains so few of these cells, compared to e.g. human peripheral blood??
I've tried to get mouse PBMC and it is just not easy. It seems like there are more rbc and it is harder to get separation of the mononuclear cells from the rbc. And remember that from a single mouse you can get perhaps 1 mL of whole blood but from a human it is easy to get 50 mL. The lymph nodes and spleens are just packed with lymphocytes. If we could easily get those from humans we'd be doing that instead of blood.
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