Dear authors, I got confused with the steps 9 to 11. During...
Dear authors,
I got confused with the steps 9 to 11. During the 7-day iNKT cell expansion, do we need to feed with aGC-loaded DC every 2 days?
I had try once with this protocol, and everything went well till the co-culture step. Then my cells didn't expand as expecte Read more Read less

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Tonya J. Webb Author Answered Oct 29, 2015
Microbiology and Immunology Department, University of Maryland School of Medicine, USA
Hi Ching-Lien,
I have found that proliferation is highly variable and the numbers that I get after expansion is donor-dependent. I get a greater yield when the donor has a high starting population (0.4-1%) of NKT cells. After 4-5 weeks, I usually freeze down aliquots. Then I take the cells out of freeze and restimulate them, and the cells will proliferate at a high level again.
The 2-mercaptoethanol is provided at a concentration of 55 mM and we use 1ml per 1L of medium.
Best wishes,
Tonya
Hi again to all,
After performing this protocol several times, I would like to know how to keep the iNKT cell growing? I got enriched iNKT population within my cellular pool (purity >95%), however, my cell counts of iNKT never reach 1 million or even more. The initial iNKT count I had was around 100K to 500K. Then I might have total cells around 1 million till week 4, and then the cell number started to decrease.
After rechecking with this protocol, I would like to know if the 2-mercaptoethanol is important for NKT growing? And it's concentration should be 5 x 10^5 M as mentioned in your protocol or 5 x 10^-5 M? I am confused.
I ever tried to re-stimulate the iNKT cells using their autologue mDC plus aGC at week 5, however, I did not still have huge amount of iNKT as I expected.
Hope you can help me out. Thank you for reading my message.
Ching-Lien
Xiangming,
It is very helpful. Thank you.
Ching-Lien
Xiangming Li Author Answered Jun 16, 2015
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
Ching-Lien,
Regarding your previous question, it is not necessary to add a-GalCer-loaded DC every 2 days. To expend iNKTs, DCs were only added one time when starting co-culture.
Ok, I will keep you posted.
Thank you.
Ching-Lien
Tonya J. Webb Author Answered Jun 15, 2015
Microbiology and Immunology Department, University of Maryland School of Medicine, USA
Dear Ching-Lien,
Let me know how your results turn out with this second experiment.
Good luck,
Tonya
Dear Tonya,
Firstly, thank you very much for your replay.
In fact, I put the purified NKT and the aGC-loaded DCs together, then I fresh complete medium+ IL-2 every 2 or 3 days. I saw many clusters on Day 2 after co-culture. However, the cell number till today (Day 9) is not increased. So I am wondering what could be the problem.
I saw very low iNKT cells presented in this donor. The purified fraction was not very convincing. Most of them looked dead from my FACS analysis. It could be a reason.
I'm trying to perform the second time. Let's see what I can get. Do you think that, instead of using CD14 beads, collecting the monocytes using adhesion ways would also work to generate the DCs?
I believe that I am going to have many questions about this expansion procedure.
Thank you again!
All the best,
Ching-Lien
Moriya Tsuji Author Answered Jun 15, 2015
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
Thank you, Tonya, whose response is absolutely correct. MT
Tonya J. Webb Author Answered Jun 15, 2015
Microbiology and Immunology Department, University of Maryland School of Medicine, USA
Dear Ching-Lien,
During the expansion you do not need to add in aGC-loaded DCs. It is a medium exchange, if your cells are rapidly dividing then you should add fresh complete media +IL-2.
Please let me know if you have any additional questions.
Best wishes,
Tonya
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