Dear authors, I got confused with the steps 9 to 11. During...

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Ching-Lien WU
Jun 15, 2015
protocol Protocol: Generation of Human iNKT Cell Lines
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
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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
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Ching-Lien WU Answered Oct 28, 2015

CEA-SRHI

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
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Ching-Lien WU Answered Jun 17, 2015

CEA-SRHI

Xiangming,

It is very helpful. Thank you.

Ching-Lien
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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.
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Ching-Lien WU Answered Jun 15, 2015

CEA-SRHI

Ok, I will keep you posted.
Thank you.

Ching-Lien
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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
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Ching-Lien WU Answered Jun 15, 2015

CEA-SRHI

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
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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
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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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