Original research article

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Aug 2012

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Generation of Human iNKT Cell Lines    

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Natural killer T (NKT) cells comprise an important immunoregulatory T cell subset and express cell surface proteins characteristic of both natural killer cells and T cells. Invariant NKT (iNKT) cells are activated by lipid antigen presented in the context of CD1d molecules, in contrast to classic T cell subsets which recognize peptide antigens presented by MHC molecules. Following activation, iNKT cells rapidly secrete large amounts of cytokines and can lyse tumor cells and virally infected cells; however, iNKT cells are reduced in patients with autoimmune disease and cancer. The potential to characterize and investigate the prospective use of iNKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human iNKT cells. In this protocol, we describe a method to generate and propagate primary human iNKT cells. Specifically, primary iNKT cells were isolated from human peripheral blood mononuclear cells (PBMC), and then expanded periodically with irradiated α-GalCer loaded autologous immature dendritic cells (DC) in the presence of human IL-2.

Materials and Reagents

  1. Blood sample to collect iNKT cells
  2. Ficoll-Paque Plus (GE Healthcare Biosciences, catalog number: 17-1440 )
  3. PE-anti-Vα24Jα18 antibody (6B11) (Biolegend, catalog number: 342904 )
  4. Anti-CD16/32 antibody (Biolegend, catalog number: 101320 )
  5. Anti-Vα24 antibody (Beckman Coulter, catalog number: IM2283 )
  6. Anti-CD3 antibody (Biolegend, catalog number: 300312 )
  7. Recombinant human GM-CSF (R&D Systems, catalog number: 215-GM )
  8. Recombinant human IL-4 (R&D Systems, catalog number: 204-IL )
  9. Recombinant human IL-2 (Proleukin) (BD Biosciences, catalog number: 354043 )
  10. Human dendritic cell isolation kit (human CD14- magnetic microbeads) (MiltenyiBiotec, catalog number: 130-050-201 )
  11. Mitomycin C (Sigma-Aldrich, catalog number: M4287 )
  12. Human iNKT cell isolation kit (Vα24Jα18- magnetic microbeads) (MiltenyiBiotec, catalog number: 130-094-842 )
  13. α-galactosylceramide (α-GalCer, KRN7000) (Enzo Life Sciences, catalog number: BML-SL232 )
  14. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875 )
  15. Non-essential vitamin solution (Life Technologies, Gibco®, catalog number: 11140-050 )
  16. MEM Vitamin solution (Life Technologies, Gibco®, catalog number: 11120-052 )
  17. Sodium Pyruvate (Life Technologies, Gibco®, catalog number: 11360-070 )
  18. 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
  19. Nalgene Freezing Container (Thermo Fisher Scientific, catalog number: 15-350-50 )
  20. Antibiotics: penicillin-streptomycin
  21. Heat inactivated fetal bovine serum
  22. MACS buffer (see Recipes)
  23. Complete medium (see Recipes)


  1. Centrifuge with swing out rotor and capable of 300-700 x g
  2. BD LSR II flow cytometer (BD Biosciences)
  3. Sterilized Pasteur pipettes
  4. 50 ml conical tubes
  5. 0.22 μM filter
  6. T-175 flask
  7. T-25 flask
  8. 37 °C 5% CO2 incubator


  1. Collect peripheral blood mononuclear cells (PBMCs). For Ficoll density gradient centrifugation separation of lymphocytes from a buffy coat or leukopheresis pack, first dilute heparinized blood with an equal volume of 1x PBS at room temperature.
  2. Add 15 ml of Ficoll (warmed to room temperature) to 50 ml conical tubes. Then slowly overlay 25 ml of the diluted blood mixture on top of the Ficoll. Centrifuge at 400 x g for 30 min at room temperature with the brake off.
  3. Carefully remove the 2-3 ml lymphocyte interface (white ring between the media and Ficoll, please see schematic diagram in Figure 1) with a sterilized Pasteur pipette and transfer to a new 50 ml conical tube.

    Figure 1. Schematic of Ficoll-Paque Plus separation of human peripheral blood

  4. Wash the cells by resuspending the cell pellets in 5 ml PBS, then combine all of the pellets into one 50 ml tube, and fill the tube with PBS to 50 ml and spin down cells at 400 x g for 10 min at room temperature with the brake on.
  5. Discard the supernatant and combine the tubes from a single individual donor to a single tube and wash the PBMCs again with 20 ml PBS. Then count the PBMCs and resuspend at a concentration of 5 x 107 cells/ml in ice-cold MACS buffer.
    Note: A leukopak is typically used for these studies and the initial volume of blood is 400-450 ml, but the product has been enriched for leukocytes and the final volume used for Ficoll separation in step 1 is around 40 ml. After the completion of step 5, it is best to continue with the rest of the protocol. If not, the cells should be cultured with complete medium at 107 cells/ml in T-175 flask overnight in 37 °C 5% CO2 incubator, and must be processed into the next step in 24 h.
  6. DAY 1- Isolate human CD14+ cells: Use human CD14- magnetic microbeads according to the manufacturer’s instructions. 
    1. For CD14+ PBMCs, culture 107 cells with 8 ml complete medium and 20 ng/ml rhGM-CSF and 20 ng/ml rhIL-4 for 4-5 days in T-25 flask to generate immature dendritic cells (DCs). Freeze the extra cells for future use at 107/vial.
    2. Keep the CD14neg fraction for iNKT cell isolation. In order to maintain the cells, culture in complete medium and 10 U/ml rhIL-2 at 107/ml in a T-175 flask. It is not necessary to replace medium when expanding DC cells.
  7. DAY 5-To expand the primary iNKT cells: Collect CD14neg PBMCs and wash with 10 ml complete medium. Use the human iNKT cell isolation kit according to the manufacturer’s instructions.
    1. To isolate Vα24+ hNKT cells:
      1. Collect CD14- PBMC and wash with wash medium once.
      2. Resuspend the pellets with 30 ml ice cold MACS buffer and pass through a 70 μm sterile cell strainer, then spin at 400 x g for 5 min.
      3. Resuspend pellets with 1 ml MACS buffer, add anti-Vα24 antibody, 5 μg/106 positive cells (NKT cells comprise 0.01%-0.5% of the lymphocyte population in PBMC). Incubate on ice for 30 min.
      4. Wash cells twice in 30 ml ice cold MACS buffer.
      5. Resuspend pellets with 1 ml MACS buffer, add anti-mouse IgG microbeads 20 μl/106 positive cells. Incubate on ice for 20 min.
      6. Wash cells once in 30 ml ice cold MACS buffer, while centrifuging prepare an MS column by adding 500 μl MACS buffer.
      7. Resuspend the cells in 500 μl ml MACS buffer. Then pipette the cells into the MS separating column. Make sure to avoid generating bubbles by pipetting slowly. Rinse the column by adding 500 μl ml MACS buffer. Repeat twice. Add 1 ml fresh MACS buffer and remove column from magnet. Place column into a 15 ml conical tube. Insert plunger and push out contents to obtain purified iNKT cells. Count NKT cell enriched fraction. You should have 0.02-0.5 million cells.
  8. In order to expand primary human iNKT cells:
    1. First, pretreat the immature DCs with mitomycin C. Resuspend immature DCs at 5 x 106 cells/ml in RPMI containing 0.05 mg/ml mitomycin C, incubate at 37 °C in the dark for 30 min, then wash three times with 10 ml complete media.
    2. Pulse immature DCs by incubating the cells in complete medium (12 well plates/1 ml per well) containing 200 ng/ml α-GalCer and 20 U/ml rhIL-2 at 2-3 X 106 cells/ml at 37 °C for 1 h.
  9. To the 12 well plates- Add 1 ml in complete medium containing 20 U/ml rhIL-2 Vα24+ human iNKT cells to wells containing α-GalCer-loaded DCs (DC:iNKT should be 5:1, for example 2 x 106 DC cells + 0.4 x 106 iNKT cells), mix gently.
  10. After 2 days, collect cells with a sterilized serological pipet and transfer into 15 ml tubes. Bring volume to 10 ml with RPMI medium containing 5% FBS. Centrifuge at 300 x g for 10 min with braking, discard supernatant, and resuspend cells with complete medium plus 20 U/ml rhIL-2 at 0.4 x 106 cells/ml, and then culture cells in 12 well plate at 3 ml/well.
  11. Culture cells for 7-10 days, and replace medium every 2 days as step 9.
  12. Flow cytometric analysis of expanding iNKT cells.
    Gate on lymphocytes and check purity by flow cytometric analysis, using PE-anti-Vα24Jα18 antibody (6B11) - see Figure 2.

    Figure 2. A. iNKT cells among human PBMCs; B. iNKT cell line isolated and expanded from human PBMCs

    Flow cytometry procedure:
    1. Collect 0.1 x 106 cells, and transfer into 1.5 ml tube, and filled with 1 ml FACS buffer (0.2% FBS in PBS).
    2. Centrifuge cell 600 x g for 5 min, and then discard supernatant.
    3. Resuspend cells in 50 μl FACS buffer, and add 0.5 μl anti-CD16/32 antibody  for 15 min to block non-specific binding, and then wash as step a.
    4. Resuspend cells in 50 μl FACS buffer, and add 5 μl PE anti-Vα24 antibody and 5 μl APC anti-CD3 antibody  for 30 min on ice in dark, and then wash as step a.
    5. Resuspend cells in 200 μl PBS, and run samples on an LSRII FACS machine.
  13. To maintain iNKT cells culture: Culture iNKT cells in complete medium plus 20 U/ml rhIL-2, and every 2-3 days replaced with fresh medium. When the density of viable cells is greater than 2 x 106 cells/ml, the cells should be diluted to 0.4 x 106 cells/ml with complete medium plus 20 U/ml rhIL-2. To prepare stocks of iNKT cells, iNKT cells with ice-cold RPMI medium plus 10% DMSO and 20% FBS at 5 x 106 cells/ml medium/frozen vial, using Nalgene Freezing Container.


  1. Complete medium
    RPMI medium 
    100 mM sodium pyruvate
    10 mM non-essential vitamin solution
    100 mM MEM Vitamin solution
    5 x 105 M 2-mercaptoethanol
    50 U/ml penicillin-streptomycin
    10% heat inactivated fetal bovine serum
  2. MACS buffer
    1 L PBS free of Ca2+ and Mg2+
    5 g BSA
    2 mmol EDTA
    sterilized by passing through 0.22 μM filter


This work was supported by National Institutes of Health (NIH), National Cancer Institute Grants K01 CA131487, R21 CA162273, and R21 CA162277 to T.J. Webb, NIH AI 70258 to M. Tsuji, the NIH AI 44129, CA 108835, and P01 AI072677 to J.P. Schneck. The method was published in Webb et al. (2012) and it is an adaptation of the methods used by Exley et al. (1997), Harada et al. (2005) and Shiratsuchi et al. (2009).


  1. Dellabona, P., Padovan, E., Casorati, G., Brockhaus, M. and Lanzavecchia, A. (1994). An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med 180(3): 1171-1176.
  2. Exley, M., Garcia, J., Balk, S. P. and Porcelli, S. (1997). Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells. J Exp Med 186(1): 109-120.
  3. Fowlkes, B. J., Kruisbeek, A. M., Ton-That, H., Weston, M. A., Coligan, J. E., Schwartz, R. H. and Pardoll, D. M. (1987). A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family. Nature 329(6136): 251-254.
  4. Harada, Y., Imataki, O., Heike, Y., Kawai, H., Shimosaka, A., Mori, S., Kami, M., Tanosaki, R., Ikarashi, Y., Iizuka, A., Yoshida, M., Wakasugi, H., Saito, S., Takaue, Y., Takei, M. and Kakizoe, T. (2005). Expansion of alpha-galactosylceramide-stimulated Valpha24+ NKT cells cultured in the absence of animal materials. J Immunother  28(4): 314-321.
  5. Prigozy, T. I., Naidenko, O., Qasba, P., Elewaut, D., Brossay, L., Khurana, A., Natori, T., Koezuka, Y., Kulkarni, A. and Kronenberg, M. (2001). Glycolipid antigen processing for presentation by CD1d molecules. Science 291(5504): 664-667.
  6. Shiratsuchi, T., Schneck, J., Kawamura, A. and Tsuji, M. (2009). Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells. J Immunol Methods 345(1-2): 49-59.
  7. Webb, T. J., Bieler, J. G., Schneck, J. P. and Oelke, M. (2009). Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells. J Immunol Methods 346(1-2): 38-44.
  8. Webb, T. J., Li, X., Giuntoli, R. L., 2nd, Lopez, P. H., Heuser, C., Schnaar, R. L., Tsuji, M., Kurts, C., Oelke, M. and Schneck, J. P. Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer. Cancer Res 72(15): 3744-3752.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Li, X., Tsuji, M., Schneck, J. and Webb, T. J. (2013). Generation of Human iNKT Cell Lines. Bio-protocol 3(6): e418. DOI: 10.21769/BioProtoc.418.

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Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
The source of human IL-2 can be E. coli. In fact, we routinely use a recombinant human IL-2 from E.coli. But it needs to be pure and without any contamination of endotoxin. I previously wrote T-75 but it is typo. T-175 is correct. Thanks.
5/18/2018 7:33:51 AM Reply
Burcu İlçe
Ankara University

Ok, thanks for all your answers. I will share the results when I apply the protocol.

All the best,

5/18/2018 10:01:12 AM

Burcu İlçe
Ankara University
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!

Dear Tonya,
I didn't ask for the 3. item, I asked for the 5th (anti-Va24 ab). When did you use this ab in the protocol? In which step? In the 4th page, 7aiii., I saw anti-Va24 but is this Beckman coulter IM2283? I thought that was anti-inkt microbeads, if not where did you use human iNKT cell isolation kit in the protocol?

Can I express myself clearly? I hope :) TIA
5/18/2018 4:04:43 AM Reply
Tonya J. Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Burcu,

Yes, you are correct. You can use the iNKT microbead kit or you can use the Va24 antibody from Beckman Coulter and then use anti-mouse IgG microbeads to enrich for NKT cells.

Thank you Moriya, for providing clarity.

Best wishes,

5/18/2018 6:55:39 AM

Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
Thanks, Tonya, for your response. Just to add, the concentration of CD14- cells is approx. 2.5 x 10^6/mL, meaning that 5 x 10^7/20 mL in T-75 flask.

Recombinant human IL-2 conversion is usually 2 x 10^4 IU/ug.

5/17/2018 11:52:20 AM Reply
Burcu İlçe
Ankara University

Thank you very much Moriya and Tonya.
Isn't the source of the cytokines that are used important? I thought it should be from human like HEK cells. As I saw you used E.coli origined IL-2, GMCSF etc. It isn't important at that point, right?

5/18/2018 1:36:41 AM

Burcu İlçe
Ankara University
Thanks a lot in advanced!
All the best,
5/8/2018 12:36:07 AM Reply
Burcu İlçe
Ankara University
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) In 8b; in previous step (8a) we plate 5x10^6 imDC/ml (means 5x10^6 imDC/per well of 12 well plate). but you want us to add gal cer and IL-2 for 2-3x10^6/ml cell? We should add for 5x10^6/ml cell, shouldn't we?
3) And finally, you gave ng/ml amount for GMCSF and IL-4 but U/ml amount for IL-2. I have 50 ug/ml IL-2 but I don't know how much to add to reach 20U/ml.
5/8/2018 12:35:13 AM Reply
Burcu İlçe
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 :)

5/8/2018 3:37:18 AM

Tonya J. Webb
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.

5/11/2018 8:44:03 AM

Tonya J. Webb
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,

5/11/2018 8:49:31 AM

Ching-Lien WU
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 expected. It may because I did not keep feeding them. Please help me to solve this problem. Thank you very much!

Best wishes,

6/15/2015 1:05:17 AM Reply
Tonya J. Webb
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,

6/15/2015 6:34:30 AM

Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA

Thank you, Tonya, whose response is absolutely correct. MT

6/15/2015 6:54:20 AM

Ching-Lien WU

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,


6/15/2015 9:56:23 AM

Tonya J. Webb
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,

6/15/2015 1:23:41 PM

Ching-Lien WU

Ok, I will keep you posted.
Thank you.


6/15/2015 2:29:34 PM

Xiangming Li
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA


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.

6/16/2015 4:01:51 PM

Ching-Lien WU


It is very helpful. Thank you.


6/17/2015 1:57:00 AM

Ching-Lien WU

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.


10/28/2015 8:50:43 AM

Tonya J. Webb
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,

10/29/2015 7:33:49 PM

Mark Exley
Useful to have the NKT protocols available here for all, thanks for posting !

Minor points:

1. to avoid any confusion, T cell medium contains not "Vitamin" but Amino-Acid solutions for those catalogue numbers (and as in ours and others published variations on these protocols):

MEM Non-Essential / Essential Amino Acids Solution, 100X

2. human NKT (like most human ab T cells) can be kept alive almost indefinitely. Some use repeated frequent stimulations (e.g. Rogers. J Immunol Methods. 2004 Feb 15;285(2):197-214), for example if rapid maximal expansion is desired, but when possible, we prefer periodic stimulations when clearly rested cultures (few if any clusters in culture) no longer proliferating effectively, to avoid changing phenotype too much with each cycle of stimulation in vitro (e.g. Exley. Eur J Immunol. 2008 Jun;38(6):1756-66).

Mark Exley
5/7/2013 1:25:42 PM Reply
Antonia Rotolo
Imperial College London
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 receive GCSF?
3. Which initial volume of peripheral blood would be desirable without leukapheresis?
4. Do you think the initial isolation of CD14 and iNKT can be omitted with an acceptable expansion efficiency?
5. How long can you keep culturing and expanding iNKT cells in your experience?
6. Can you see any difference in the expansion efficiency among donors?
Many thanks in advance for your help! I am very grateful indeed!
Best regards,
Antonia Rotolo

4/9/2013 1:08:28 AM Reply
Tonya J. Webb
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

4/9/2013 3:14:32 PM

Antonia Rotolo
Imperial College London

Again thank you very much!

4/9/2013 3:31:15 PM

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