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Conventional DC preparation from Human Monocytes and co-culture with autologous T-cells.

I am preparing DC from Human Monocytes which I isolated from PBMC (using ficoll gradient) using Miltiyeni CD14 micro-beads isolation. Then I culture them in 6 well plate in presence of GmCSF n IL-4 50ng/ml. After 7 days I have to co-culture these DC with autologous T cells. Now the problem I am facing is how to detach these adhered DCs? What amount and when should I add IL-2 after co-culturing them with autologous T-cells?

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  • James George · University of Alabama at Birmingham
    If you chelate the Ca and Mg, they will tend to pop off the plate. Quite often we will use HBSS or PBS with a small amount of EDTA to do this.
  • Daniela Tomasoni · Ospedale di San Raffaele Istituto di Ricovero e Cura a Carattere Scientifico
    Hi Raghav,
    are you using FBS or human serum in the colture media for your DCs prep?
    For my preparation I use RPMI 1640 supplemented with FBS and I do not harvest the cells that adhere to the plastic.
  • Elisabetta Volpe · Foundation Santa Lucia
    Differentiated DC do not adhere to the plastic....you can remove them easily with a pipette. Then you gently add DC on the top of T cell culture. CD4 T cells will produce enough IL-2 their self during the coculture, it is not necessary to add exogenous IL-2. However, in case you perform autologous coculture you must add an antigen or the antiCD3/28 to have a T cell response.ciao
  • Urban Švajger · Blood transfusion centre of Slovenia - Zavod Republike Slovenije za transfuzijsko medicino
    Like Daniela said, when using RPMI+FBS, fully differentiated DCs are in suspension and you don't need to detach. However, when using serum-free media with/without addition of e.g. human AB serum, the DCs tend to adhere quite firmly. A good trick is to wash the wells with Ca and Mg-free PBS or DPBS at least 2 times (during this procedure, more cells can already be detached immediately if washing with 1000ul pipette tips than with serum pippette, that washes more gently). After 2 washes, if DCs still remain attached, add some Ca, Mg-free PBS and place the cells at +4 C for approximately 20 minutes. Take them out of the fridge and repeat the washing with 1000ul tips. Surely, most of the cells will detach.... good method to avoid trypsinization, try it.
  • Felix Lichtenegger · Ludwig-Maximilian-University of Munich
    Hi Raghav, it is not completely clear to me if it is the whole DC generation protocol that you are describing. Am I correct that you want to gain immature dendritic cells? In fact, these tend to be slightly adherent, even after six days in culture, but you should be able to remove them from the plate with a little bit of scratching. I agree with Urban that use of cold PBS is a good method to remove them more easily. But if you rather want to have mature DCs, like most people do, and add a maturation cocktail for the last 24 to 48 hours of DC generation, then the fully matured DCs should certainly be in suspension.
  • Eiman Elwakeel · Alexandria University
    Dendritic cells are barely attached to the bottom of the plate, So just using cold Ca, Mg free PBS for multiple washing is helpful. I think you won't need to add EDTA . One more thing, use a Pasteur pippet for washing, it's better than the micropippet tips (less vigorous on cells).
  • Eiman Elwakeel · Alexandria University
    Plus I don't think that adding IL2 is essential here!
  • Carolina Behrens · Pontificia Universidad Católica de Valparaíso
    Hi, when I generate DC from PBMC I use a cell scraper to harvest the cells, you must be carefull and gentile with the cells to mantain them alive, you can use PBS 1x or RPMI 10% to do that, and in my experience I don't have problems with that, or you can harvest the DC using buffer PBS 1x + EDTA 2mM. Now, when I make the co-culture with T cells I add IL-2 at 150-200 U/ml at day 1,3,5, and at seventh day I collect the T cells for my experiments. I hope I've helpfull.
  • Nhan Npt · Chonnam National University
    Hi, Here is my experience!
    When we used any media (with or without serum or FBS), after 6 or 7 days culture with GM-CSF and IL-4, the differentiated DCs will be fully differentiated DCs and floating in suspension. In any of the functional assays, we do not harvest the adherent cells (whenever present). Some papers showed that the adherent cells from DC culture plates are regulatory DCs or macrophage-like cells. these cells are not stimulatory and may be suppressive.
  • Priya Ramanathan · Cancer Institute (WIA)
    Hi
    IL4 needs to be in the medium if you have not matured them or they wil become macrophages and stick so keep the cocktail replenished you wont have the problem.
  • Francesca Urbani · Istituto Superiore di Sanità
    It' s not clear (to me) which functional test you want to perform with autologous T cells. Maybe, adding of IL2 depends on the type of test...
    Regarding the first question, i agree with Urban.
  • Raghav Oberoi · Hannover Medical School
    Hi Thanks everyone for your suggestions. I am using serum free X-VIVO 15 media from Lonza. After 7 days culturing them with GMCSF n IL-4 , after this I stimulate them with oxLDL (oxidised low density lipids) , nLDL (native low density lipids) and TNF alpha. I will stimulate them for 24hr then wash them n co-culture with autologous T-cells. Do I need to irradiate the stimulators i.e the DC prior to co-culture with T-cells. I have seen some papers in which they irradiate and then co-culture them.

    Thanks for all the help.
    Cheer's
    Raghav
  • Cecília Favali · University of Brasília
    Hi Raghav,

    I agree that immature DCs do not adhere to the plate. You just need to wash the plate about three times to obtain them after seven days. In fact I remove 200 ul per well of medium and add 250 ul of fresh cytokine supplemented medium in day 3 and 6 during culture. This is a precious tip! For the co-culture I used to irradiate to perform a mixed limphocyte reaction but when I screened for autologous T- cell derived IFN-g DCs were not irradiated. DCs should be as viable as possible. I had a low backgroud in the negative control. You can use anti-CD28+anti-CD3 as positive controls for your proliferation assay. In my hands, the best time point for proliferation was 96 hours.
  • Nhan Npt · Chonnam National University
    Hi, Raghav,
    When I cocultured DCs with autologous DCs, I don't need to irradiate the Dcs. I only irradiate the DCs when I used mouse DCs. In case of human DCs, I think we don't need to irradiate them. This is my background. The time to stimulate autologous DCs depend on the purpose of experiment. In case of CTL generation, we usually restimulate with same DCs at day 10-12, and harvest the CTLs at day 20-22. In case of proliferation assay, we usually coculture 3-5 day.
  • Deleted
    I don't think that adding IL2 is essential for adherence. incabation for 2hours is a 37ºC/5%CO2 is suffficient.
  • Dario Leone · Medical University of Vienna
    I strongly suggest to use only autologous serum instead of FCS, FBS to avoid non-specific antigen presentation. Plus the dendritic cell are very sensitive cells and pipette them too hard or scraped away or irradiate them or add cold medium into a 37° culture make them respond to the shock. My suggestion is to try to minimize the manipulation. For harvest just cut a 1 ml tip and very gently resuspend them, avoid bubble, avoid change of temperature in both ways and do not centrifugate them too much. For the T cell co-culture, try first with PHA, it need presentation and in 3 days you can already see proliferation.
  • Mohammad-Ali Shahbazi · University of Helsinki
    Hi, Is it possible for you to explain me how i can coculture monocytes (THP1 cells) with T cells (Jurkat). I am planning to coculture them to evaluate the effect of some nanoparticles on the activation of monocytes and T cells by measuring cytokines
    Thanks

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