Dear all, I would like to thank you for your protocol. I much...

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Antonia Rotolo
Apr 9, 2013
protocol Protocol: Generation of Human iNKT Cell Lines
Dear all,
I would like to thank you for your protocol. I much appreciated it!
I am a PhD student at Imperial College London.
I hope you don't mind if I ask for some questions.
1. Do you collect samples from healthy donors?
2. Before leukapheresis, do donors receiv
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Antonia Rotolo Answered Apr 9, 2013

Imperial College London

Again thank you very much!
Antonia
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Tonya J. Webb Author Answered Apr 9, 2013

Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Antonia,

Please see below for a point by point response to your questions:
1. Yes, we collect blood from healthy donors.
2. No, the donors do not need to received G-CSF.
3. Without leukapheresis, the minimum amount of blood that I have used is from 2 CPT Vacutainer tubes (16-18ml).
4. If the initial isolation is skipped then the expansion will be minimal unless the donor has a high percentage of circulating NKT cells (>1% of the total T cell population).
5. I can usually keep culturing and expanding for NKT cells 5-6 weeks, but some lines have been maintained for ~3 months without thawing a fresh aliquot.
6. Yes, there is a high amount of variability between the donors, usually the higher the initial population of NKT cells the higher the expansion efficiency.

Good luck with your studies and please let me know if you have any additional questions.

Kind regards,
Tonya Webb
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