Dear colleaguge
At present i m working on certain Immunology based experiments, and i m planning to isolate and purify the thymoctes from human thymus which i will use further as a antigen for injecting into the animal to develop certain antibodies
In order to isolate thymocytes, you first need to eliminate fat tissue as much as possible. This can be done in a perti dish (10cm) containing PBS, using a scalpel, under a stereo-microscope (5 to 10 fold magnification. Then you disrupt thymic tissue by gently pressing through a 100µm sterile mesh in a petri dish containing PBS or culture medium. Thymocytes can then be FACS analysed using CD4 and CD8 labelling to confirm the presence of DP cells (about 80%).
Dear Dr. Remi, Than q so much for your suggestion, I applied your protocol ,It helped me a lot in my bench work, and I am getting good number of cells from human thymus, I am using PBS buffer+0.25% BSA+0.2mM EDTA for isolation of thymocytes as a media, but I faced some trouble like after isolating the cells , I am observing the cell viability by using 0.08% of tryphan blue with in 2-3 mins,, but I am observing all are dead cells (blue spots) but all cells are looking morphologicallly healthy, active, and intact (without tryphan blue) under microscope,, Sir can u suggest me , is it ok for injecting this dead thymocytes in to horse or rabbit for development antibodies against human thymocytes????, In addition to that, I m preseving the isolated thymocytes in a -20 degree freezer with cryopreservative like DMSO (10%), but I am observing after 2 days , all the cells are getting lysis and certain gelly things are floating , may i know why its happening like that? what is the best method for storing and human thymocytes for longer period?
Hi,
I'm quit surprised by your observations.
You should perform the isolation in culture medium (or PBS but Culture medium is better), with 10% FCS, not with BSA and EDTA.
Using Trypan blue, dead cells do not only contain blue spots, they are totally blue
-20°C is not cold enough for cell conservation, you should at least store them at -80°. The best conservation medium is 90%FCS 10%DMSO, freezing should be quite slow. For that, put the vials in a polystyrene box full of absorbant paper and put the box at -80°C for >24 hours before transferring to liquid nitrogen. In contrast, thawing the cells should be a rapid process. Shake the frozen tube in a 37°C water bath and immediately dilute 10x in culture medium before pelleting the cells, then wash the cells before further experiment.
Concerning your immunisation protocole, I have no experience in that domain but I guess that intact living cells (or fixed cells) should be better that dead cells. You will need to perform at least 1-2 boosts.
Dear Dr, Remi, Than q so much for your valuable suggestion, is it possibile to get your email id, I need to communicate with you through mail about my result part for some further clarification
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