Preparation of single cell suspension from spleen and thymus

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BACKGROUND

Spleen and thymus are the lymphoid organs of an animal starting from lower vertebrate like fish to higher vertebrate like human. Therefore, culture of cells derived from these lymphoid organs will help to understand many aspects in the field of immunology.¹

Aim: Aim of this experiment is to isolate single cell suspension from the spleen and thymus of the mouse.

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REQUIREMENTS

Tissue sample:  Spleen and thymus from mouse

Apparatus:     Sterile petri dish

Syringe – 5 ml

Centrifuge tubes – 15 ml

Cell strainer – 70 µM

Instrument:     Swinging bucket rotor centrifuge
Chemicals:      1X HBSS w/ 0.1% BSA + 0.6% Na-citrate (without Ca²⁺ and Mg²⁺)

Antibiotics/ Antimycotics

Ammonium chloride solution – 0.17 M

Collagenase type ΙΙ

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PROCEDURE

Firstly, mix HBBS with antibiotics/antimycotics (1:100) keep ready for the experiment. Place the tissue (freshly dissected mouse spleen and thymus) in the petri dish, chop it, and mince it quickly as fine as you can. Add collagenase type ΙΙ (1 mg/ml) to digest fibrotic tissue. Add 5 ml 1X PBS and mince the tissue with the syringe plunger, after fine mincing, place the cell strainer in the centrifuge tube, use the syringe plunger to macerate the cells through the filter add 5 ml HBSS. Centrifuge the tube at 300 × g at 4⁰C for 5 min. Remove supernatant and add HBSS solution to wash again. Add 10 ml of 0.17M ammonium chloride solution centrifuge 300× g, discard supernatant add HBSS solution and go for further analysis.^2,3

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CONCLUSION

The single cell suspension from mouse spleen and thymus cells are ready for culture.

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REFERENCES

1. Mallone R., et al. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T-cell responses:
   position statement of the T-cell workshop committee of the immunology of diabetes society. Clinical and experimental immunology. 2010.
   
2. Thermofisherscientific.com
   
3. Protocols from stem cell technologies. Available at stemcell.com.