Hello,

Use fetal thymus organ culture

http://cshprotocols.cshlp.org/content/2007/8/pdb.prot4808.full.

so thanks Dear shiny Titus for answer me.

with best wishes

but PDF in my internet host network can not be download?please if do you have access to this PDF, send me.

Thanks.my email address is : [email protected]

thanks. your generous guide.

Which animal species you are using for total thymus organ culture? Fetal thymus will not be full representative, as thymus is peak in growth till the puberty of any animal species. And age of puberty differs in different animal species, hence you should know which animal species you wish to culture whole thymus organ culture. I have experience of handling chicken and goat whole thymus organ and thymic cells culture successfully. So, if you wish I can provide the protocol for that.

If you can separate 2 intact lobes of thymus from the body of freshly killed mouse, I think it is not a big deal to prepare the whole thymic cells.

For culture purposes, the thymus has to be collected from a freshly killed animal, so that live thymocytes count is maximized and also vital cell structures are not lost.

I have experience of separating goat thymus from a young goat and also chickens. The thymus in these animals and birds are situated as thymic lobes ventrolaterally along the trachea, which can be dissected out using sterile forceps carefully without cutting through the lobes, to avoid the loss of thymic cells in the dead-body itself.

So, you should know how to collect the mouse thymic lobes, if you know the exact location and identify the thymic lobes in a freshly killed mouse. Further, treat the tissues, as under :

1. Under strict sterile conditions in a Laminar Flow, using blunt end forceps gently remove the thymus lobes out of the freshly killed mouse body.

2. Transfer the lobes in Ca2+Mg2+ free PBS, pH 7.2, in a sterile petriplate. Give thorough washings with 2-3 changes of sterile PBS.

3. Using sterile small scissor (pointed ends), cut through the thymus lobes in the petriplate itself, finally tease the smaller cut tissues thoroughly to ensure that all the thymic cells are separated out of the thymic lobules.

4. Add about 5 ml of sterile PBS, and filter through a sterile muslin clothed sieve to remove the course tissue.

5. Collect the sieved cell suspension in a centrifuge tube, and spin at 800 rpm for 10 mts to remove any platelet contamination, if at all.

6. Give 2 more washings with nearly 5-10 ml of PBS at 800 rpm for 10 mts each.

7. Discard the supernatant and resuspend the sedimented thymocytes in about 5 ml of PBS, pH 7.2.

8. Do live cell count using trypan blue stain, as you do for usual lymphocyte count.

9. Adjust the cell concentration (usual 1-2 million per ml) as per your experimental need and use for further experiments.

 I did not follow any specific protocol, and used my individual experience and skill based on my subject knowledge to handle total thymocyte counting, culture and use for immunization experiment. Thymus lobes and lobules are very soft and fragile tissues, and can be easily cut through using pointed scissor and if further teased, most of the thymocytes are obtained.

 If you want to use thymic nurse cells, epithelial cells, and stromal cells, you may also check the supernatant after first centrifugation that you are not losing any cell of use to your experiment.

 If you want to use most of the thymic tissues fractions, you may also use whole thymic tissue after thorough teasing as such, and monitor progress of your experiments. Finally, you will be able to standardize your experiment, as per your need.

If you have any more queries, please feel free to ask. I will be too glad to help out of my experience.

Very Thanks for your answer my Freind. For give thymic nurse cells, epithelial cells, and stromal cells,Do I need to any enzymatic digestion for example tripsin or collagenase?

with best wishes.