Isolating lymphocytes from a mouse spleen involves several steps:

1. Spleen collection: Harvest the spleen from a mouse and place it in a sterile container with cold PBS (phosphate-buffered saline).

2. Spleen dissociation: Mechanically dissociate the spleen into a single-cell suspension using a tissue grinder or a syringe.

3. Red blood cell lysis: Remove red blood cells by adding a lysis buffer (e.g., ACK lysis buffer) and incubating for 5-10 minutes.

4. Centrifugation: Centrifuge the cell suspension at 300-400 x g for 5-10 minutes to pellet the cells.

5. Cell washing: Wash the cells with cold PBS to remove any remaining debris.

6. Density gradient centrifugation: Layer the cell suspension over a density gradient medium (e.g., Ficoll-Paque) and centrifuge at 400-600 x g for 20-30 minutes.

- Spleen dissociation using a cell strainer and the back of a syringe works well.

- RBC lysis is good for better FACS analysis immediately after dissociation; however, not required for in vitro culture (the red blood cells will die)

- There's no need for density gradient centrifugation since after several days, B cells and most myeloid cells will also die.

- If you want to isolate only T cells, using a magnetic enrichment step is fast and easy (anti-CD3)

Read Tlymphocyte response to ceacal coccidiosis

Paul Kaze Joao G Magalhaes Harald Kühnel

Dear all, I appreciate your answers and time.

Based on your kind answers, I didn't find any big difference between the protocols you mentioned and mine. I mean, I almost do what you mentioned about dissociation, cell strainer. What I just didn't do was the final density gradient centrifugation. Nevertheless, my cells die in this condition.

The main questionable problem is: why did more than 90% of the cells just after one day undergo apoptosis and die? and how can I avoid it?

Try a Complete RPMI Medium:

RPMI-1640 supplemented with:

10% FBS

1% GlutaMAX

1% non-essential amino acids

10 mM HEPES

1 mM sodium pyruvate

1% penicillin/streptomycin

50 µM β-mercaptoethanol

Try to add IL-2 at 50 U/mL to improve T cell survival and proliferation.

+/- PMA/ionomycin or CD3/CD28 to also enhance T cell survival and function. (positive control of T cell activation and proliferation)

Maintain cells at an appropriate density (around 1 million cells/mL) to ensure optimal growth conditions. Use rond bottom 96 wells plates

If you have already implemented these steps and continue to experience issues, consider evaluating the quality of your reagents, especially FBS, as batch variations impact cell survival.

Best, Joao

Without any stimulation primary lymphocytes normally will start dying, even if 90% after day 1 might be a bit fast. You'll need to stimulate, as the others say, either through CD3-CD28, or through antigenic peptide, or PMA/ionomycin. That also depends on the mouse strain/genetically modified line you're using. Hope that helps.

Use a gentle macs dissociator with "C" tubes to break up the tissue and cells without damaging lymphs. Pass liquid thru a 40 uM falcon strainer, discard stainer. Then centrifuge gently at 350 to 500 x G to pellet the Leukocytes. Resuspend in staining buffer (for FCM) or RPMI. best wishes