Dear authors,I have some questions with the protocol.1) In 6b,...

Burcu İlçe
May 8, 2018
protocol Protocol: Generation of Human iNKT Cell Lines
Dear authors,

I have some questions with the protocol.
1) In 6b, you say culture cells (CD14neg) in T-175 flask at 10^7/ml. It means app. 50x10^7 cells in a flask right? This is for day-1. Than you go with step 7 (day 5). Will we go on with the cells in T-175 flask?
2
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Tonya J. Webb Author Answered May 11, 2018

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

Hi Burcu,

You can purchase the iNKT mAb (clone 6B11) from several companies- Biolegend, BD, or, Miltenyi Biotec. It is used to check the purity of your NKT cell population by flow cytometry.

I hope that this helps!

Good luck,
TJW
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Tonya J. Webb Author Answered May 11, 2018

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

Dear Burcu,

1) Regarding the number of cells (10^7) is 10 million cells; step 5 states 50 million cells.
- Each step is a continuation from the previous step.
2) In 8a, you will treat 5 million cells/ml with mitomycin C in a conical tube.
In 8b, after washing, 2-3 million DCs will be pulsed with antigen at the indicated concentration, and then NKT cells will be added to the plates.
3) The company that you purchased the IL-2 should also have the concentration in international units (IU). I am not sure about the conversion.
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Burcu İlçe Answered May 8, 2018

Ankara University

And one more question :)
4) You listed materials for the procedure. I didn't see the useage of 5th item; Anti-Va24 antibody. Where and when will we use it? Thanks again :)
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