I am working on a experiment to analyze the immune synapse between T cells and macrophages. In our protocol to stain the cells with fluorescent markers, we have quite a few pipetting, washing and centrifugation at 300G steps, before and after fixation.
I am wondering how many immune synapses get disrupted by all these mechanical forces. Does anybody have an idea how strong and rigid this complex is?
Thanks in advance!
Hi Leroy,
I have not worked with immune synapses. However, I have used mature neuronal cells that projected between different wells. I can highly recommend you to make the rinses pretty gently. The fixation step is really important, you do not hesitate to make some testings on there.
Hope you can obtain great results,
Jordi.
Dear Leroy,
Please find attached some papers that hopefully will help you with your questions. I am not exactly sure about the conditions of your experiment and your readout, but especially the Hämmerling paper gives a good idea about the strength of the immunological synapse in a system with APC - T cell co-culture.
Article Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse
http://www.pnas.org/content/pnas/106/42/17852.full.pdf
I hope that helps!
All the best,
Michael
The immunological synopses are quite stong due to the reinforcement of the cell-cell interactions by additional surface molecuels and the cytoskeletal reorganisation. However, it depends on the timing of the sample processing. The following article might help you with more technical details for you experiment. In my opinion, gentle washes may not desrupt the synopses. Good luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332248/?report=reader
Immunological synapses are usually strong binding and do not disrupt by gentle washing, however the pH and the quantity of tween in washing buffer is important here which can disrupt after certain limit.
I am not exactly sure about the conditions of your experiment and your readout, but especial most of the time the pH, temperature, concentration and the quantity washing buffer are important things which can disrupt after certain limit. so in my opinion it may be better to check those things and standardize the work protocol. Good luck
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