What type of DC you have the intension to investigate. DC derived from CD34 + GM TNF, or of monocytes GM + IL-4; a priori it would be DC from monocytes as you study the CD68 marker, which is the marker of macrophages. In this case we must follow the CD1a, CD208 and CD209 which are specific to the maturation of monocytes into DC.

However it is more reliable to monitor DC-T interaction with DCs derived from CD34 + GM TNF, because most representative of the various populations of DCs that can be found in vivo. In this option, you must follow the CD205, CD206, CD207, CD209, CD303 and CD304.

If you maintain your choice of MonoDC, beware of contaminating cells: basophils, MDDCs and pDCs that can interfere in your study

For information ; using a ratio of 1 DC cell to 10 T cells; As for me I prefer the flat bottom wells, we are best seen the cooperation of different cell populations.

Are you using cell lines or primary cells? I never used cell lines, so my advices are for human primary cultures.

If you are doing allogeneic co-culture, no need to add IL-2 to the medium. It is better to use a serum-supplemented media. I usually use RPMI with 10% fetal bovine serum. The U-bottom 96 well plate is, in my opinion, the best choice for the co-culture, because this forces the cells to interact, boosting T cell proliferation. In the beginning of the co-culture, you can see only one place with cells in the bottom of the well, but after some days, they start to migrate and you start to see a lot of different white points in the bottom of the well.

I usually have no problem to distinguish T cells from DCs by using FSC-SSC profile, even though I use CD3 to confirm my population. I never saw CD3 loss in the co-culture, only when activating T cells with PMA. I don’t know what could be happening. I would suggest finding another DC marker

What type of DC you have the intension to investigate. DC derived from CD34 + GM TNF, or of monocytes GM + IL-4; a priori it would be DC from monocytes as you study the CD68 marker, which is the marker of macrophages. In this case we must follow the CD1a, CD208 and CD209 which are specific to the maturation of monocytes into DC.

However it is more reliable to monitor DC-T interaction with DCs derived from CD34 + GM TNF, because most representative of the various populations of DCs that can be found in vivo. In this option, you must follow the CD205, CD206, CD207, CD209, CD303 and CD304.

If you maintain your choice of MonoDC, beware of contaminating cells: basophils, MDDCs and pDCs that can interfere in your study

For information ; using a ratio of 1 DC cell to 10 T cells; As for me I prefer the flat bottom wells, we are best seen the cooperation of different cell populations.

Please have a look at:

Eur J Immunol. 2014 Dec;44(12):3543-59. doi: 10.1002/eji.201444477. Epub 2014 Oct 27.

Concurrent interaction of DCs with CD4(+) and CD8(+) T cells improves secondary CTL expansion: It takes three to tango.

Hoyer S1, Prommersberger S, Pfeiffer IA, Schuler-Thurner B, Schuler G, Dörrie J, Schaft N.

There you can find co-culture conditions and possibilities to distinguish T cells from DC (FSC/SSC or CD3/CD83 staining).

Hi,

Thank you all for some very helpful input! Particularly Ceri, the cell labelling idea is extremely helpful! I should have thought of this - a fresh pair of eyes is always useful! 

My co-cultures are between immortalized primary murine cell line with autologous T cells.

One last question I have remaining is how long you culture - in terms of how long Dcs should be in culture in isolation in order to produce sufficient IL-2 (usually plated at 2x10-6 in T75's) to sustain T cells, and how long to culture the two together.

I will look through the journal now, thanks very much!

Best and many thanks

Brittany

Hi 

I have question? 

You have T cells line. What about Dendritic cells?  Dose it isolate or dose it generate? 

DC culture needs to autologouts  serum. but when you have co culture its different.

Please look at MLR technical guide.

for sorting: DCs and T cells are fully different in FSC and SSC. So with out using Ab, you can distinguished them. 

please look at whole blood pattern in FSC and SSC for site of T cells and then use articles for site of DCs in dot blot (flowcytometry) pattern. 

For expression of CD3 and CD68... use MFI character before and after co culture. I think its better for identify and solving a problem.

Finally, observation is important level before any action. dendritic cells in best formation are dendrite non-adherent or semi-adherent cells in culture.

I think T-25 flask is better for your culture and please use adherence flask.

Lose CD3 and CD68 maybe its related to cells viability. please check the cells viability in this stage. 

good luck.

Thanks Babak. I am using cell lines, for both DC and T cells. There is no isolation from blood. These two cell lines do not show a difference in FSC and SSC. Ideally, I am looking for a method which allows me to maintain the cells in culture after isolating DCs from T cells.

Best wishes

Brittany

Brittany I can't understan!!!!  Are sure about FSC and SSC?

How about Ag? Do you have Ag in cell culture process? 

DCs+ Ag, active T cells and following that secretion of Il-2.

I suggest to use DC in 6 well or 12 well plates rather than flasks. Label the T cells with CFSE before co-culture. You can check the surface expression by a CD3/CD11b expression. After labeling T cells, co-culture the cells in round-bottom 96 well plates. This ensures good contact between the cells. Or you can first see that DC are covering the wells completely (6 or 12 well plates is fine) and then add T cells.

FSC & SSC could be similar for cell lines. T cell lines tend to be much bigger than classical T cells thus matching DC in size.

CFSE labelling is good but if your cell line is proliferating fast, you may loose CFSE before the output point of your experiment. 

I would suggest culturing T & DC in round bottom plates to optimize interaction as has been stated before. Typical DC 1:5 T ratio is best. If proliferation is expected, 10,000 DC + 50,000 T are enough to start. 

If DC are to received stimulation, stimulate them 24h prior adding T cells. After T cell addition, you can follow the co-culture up to 7-10 days with IL-2 supplementation every 3 days (for primary cells, proliferating cell lines may require more). 10ng/ml should make it. 

T cells never really "loose" CD3, but CD3 as CD4 can be downregulated following activation. CD5 and CD2 are good alternatives for T cell markers. For your DCs, MHCII is best. 

If you need to isolate the cells later, magnetic sorting is your best (or only) option if you don't have access to a cell sorter. I would suggest Stemcell tech rather than Miltenyi as Miltenyi column tends to stress/activate cells, especially myeloid. 

Hi All,

I just wanted to reply to say thanks for all of your useful input on this topic. The original plans were pushed back so we are moving towards this aspect now, and all of your advice has been very helpful!

Many thanks

Brittany