How to isolate naïve CD4+ mouse T lymphocytes?

Tips & Guidelines
Inès F.
By Inès F.    time to read 5 min

We all know that a better understanding of immunology has enabled many improvements in disease treatments. So naturally, you want to profit from this trend. To do so, you need to know the best way to isolate your naïve CD4+ T lymphocytes.
Cell isolation is a critical step which must be carried out before the differentiation steps which lead to experiments. Due to the central role of isolation, researchers (like you) have already studied the process to find ways to improve our understanding of diseases – and that’s why the following paragraphs can give you some helpful advice.

What is a naïve CD4+ T lymphocyte?

The soldiers of our immune system

We know that CD4+ T cells help destroy pathogenic agents by activating other immune system cells, like CD8+ T cells and B cells. They are called “naïve” when they have never been stimulated by a pathogenic agent.
Nowadays, at least seven CD4+ T cells subsets are known to exist. They differ in their cytokinic secretion profiles and their functions: Th1, Th2, Th9, Th17, Th22, TFH (follicular helper T cell) and iTreg (induced-regulatory T cell) (O’Shea, J. J. and W. E. Paul, 2010).

The maturation of CD4+ T cells

Naïve CD4+ T lymphocytes come from hematopoietic stem cells. The maturation of these stem cells into naïve CD4+ T lymphocytes begins in the bone marrow. From there, immature T cells migrate to the thymus, where they continue their maturation. The thymus and the bone marrow are primary lymphoid organs and from there, cells migrate to secondary lymphoid organs (spleen and lymph nodes) via the blood. Once in the secondary organs, they can be activated if they meet certain specific antigens.

Schematic representation of naïve CD4+ T lymphocyte maturation
Schematic representation of naïve CD4+ T lymphocyte maturation

Isolation of naïve CD4+ T cells

Step 1: Harvest of the spleen and lymph nodes from mouse

Where can we find your cells of interest?

For the dissection, you should choose a euthanasia method which prevents the animal from feeling any pain (the choice does not affect your assay in any way).
To do the isolation of the naïve CD4+ T cells, you must recover secondary lymphoid organs, i.e. the spleen and lymph nodes. You will find the spleen under the stomach and above the intestine on the right when you look at the mouse. The lymph nodes are more complicated to get out! You can find some of them just above the bladder (around the uterus, in a female), under the foreleg shoulder joints and in the neck.

Schematic representation of fresh spleen and lymph node extraction
Schematic representation of fresh spleen and lymph node extraction

All these freshly recovered organs are placed in a dish containing the complete medium. Then the spleen and lymph nodes are physically separated.

Step 2: Cell separation using a Miltenyi Biotec Kit

How to recover only my naïve CD4+ T lymphocytes?

Fine, you have accomplished the first stage! The next step consists in an isolation of naïve CD4+ T by using a two-step selection protocol, following Miltenyi Biotec recommendations.

  • Firstly, we want to separate the cells which express CD4 from cells which don’t. To do this, we carry out an indirect magnetic labeling. The column is used to trap all the cells which don’t express CD4 with a biotin-antibody cocktail.

  • Secondly, we want to recover only naïve CD4+ T cells among the pool of CD4+ T cells. So, we do a direct magnetic labeling of CD4+ T cells with CD62L Microbeads. We use CD62L microbeads because naïve CD4+ T cells express a high level of this marker and lose the marker once they meet an antigen.
    After elution of the fraction of interest, you can count your cells.

    Schematic representation of indirect magnetic labeling in cell separation

    After these steps, you can say “I did it!” Bravo!
    But now, how about differentiating them? You cannot leave the cells like that! Have a look at our other blog post “Differentiate your naïve CD4+ T lymphocyte into Th17” – you will certainly find what you need.
    But if you are tired and you would prefer to work on your cells later, you can simply freeze them. I suggest you read our other blog post, which can guide you in your freezing manipulation. Take it easy…

    Guidelines for naïves CD4+ T lymphocytes isolation

    Technical note

    Download protocol