I isolated the thymocytes from human thymus for development antibodies against thymocytes by mechanical distruption only (no enzyme treatment) by using PBS, but i realized that, less number of cells are recovering, further cells are getting lysed after a period of time in PBS. can anybody suggest me, how to maintain the thymocytes after isolation? which kind of media i need to use? is it ok if i use RPMI or DMEM with FBS? what is the best method to isolate huge number of thymocytes from human thymus? can we use trypsin for isolation of thymocytes instead of collagenase?what is the storage condition of thymocytes after isolation? is it necessary to use DMSO or glycerol before store in cooling condition? how much quantity of thymocytes required to inject in horse for the development of antithymocytes antibodies? and how many days we need to inject?
actually am totally new this field, this work is highly essential for continue my research work,its my request to all my collaegue, if any body explain all my doubts in a descriptive manner, it will be highly beneficial for me
Dear Sudhansu,
I have no direct experience with thymocytes but i have some with limphocytes, and since thymocytes are lymphoid cells, maybe my comments could help. I kept peripheral blood lymphocytes for 24 hours at 37°C in RPMI suplemented with L-glutamine and Fetal Bovine Serum. The cells were treated with gamma radiation up to 5 Gy (relatively high dose) and I recovered enough cells to further experiments. Perhaps using a similar medium could inprove your cell survival. Cool storage down to 4°C is possible without adding cryoprotectants, but each cell type is different to each other.
On the other hand, I am not sure if injecting whole cells is a good idea to obtain antibodies. Whole cells would trigger a cellular immune response in the host organism, mediated by CD8+ lymphocites and innate immune cells like eosinophils and NK cells. Also complement system activation would be involved in the response. Of course that antibodies would be also produced, but in a lesser and messier way, I believe.
Usually, a protein (or a protein extract) is injected to host animal (horse, rabbit, goat, mouse, etc). If you want to use those antibodies to further label and identify thymocytes, you should try to find a distinctive protein or proteins for these cells, and then try to obtain them in the cleanest or purest possible form (which is not necesarily a pure or near-to-pure extract).
Hope my comments are of some use to you. Good luck.
Than q, Dear Manueal for sharing all valuable information,
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