23 replies to this topic

[Astarte Biologics]

Hello Friends,

I have just entered into immunology research and I need to know the following: Please try to help me by answering the following questions if you have expertise.

1. What's the reason behind using isotype controls in an experiment? I need the mechanism behind the usage of isotype controls in experiments (often when we go for flow cytometry)?

2. What is the principle of using IL-4 and GM-CSF to generate DCs from monocytes (i.e. for generating MDDCs)? I understand IL-4 is an anti-inflammatory Th2 cytokine!

3. What does PolyI:C do to make DC mature?

Please help.

Thanks
Shankar

1. The reason for using isotype controls in a flow cytometry experiment (or any other experiment for that matter) is to control for any non-specific binding of the immunoglobulin to the cells. There can be significant binding of immunoglobulin to macrophages, monocytes or granulocytes for example because of the expression of receptors for the Fc portion of the molecule. Even if you don't have that kind of binding there is some degree of sticking on any cell type. If you compare cells that are completely unstained to those that are exposed to isotype control you'll see that there's a difference.
2. Good question. It works but I'm not sure anyone really knows why or how. Some say the IL4 suppresses CD14 expression but others say that GM-CSF is enough. I think of monocytes as a non-activated myeloid cell that can differentiate to macrophage or DC depending on the stimulus.
3. PolyI:C activates DC through a TLR. Can't remember which one - TLR9 I think. This mechanism probably arose as a way of responding to viruses via the presence of nucleic acid.

Hope all that is helpful.

www.astartebio.com

[miBunny]

Atarte answered question 1 so beautifully there is nothing more to add.

IL-4 is not necessary when generating DCs from murine bone marrow but it important for generating human DCs. The generation of DCs is really an induced developmental process. What we are really doing is taking early progenitor cells and sending them down a developmental pathway by culturing the cells with a bunch of cytokines. The progenitors of interest in this case have the ability to become either DCs or macs. IL-4 is thought to be acting on the human progenitors to suppress the development of the mac lineage and to enforce the DC developmental decision.


Poly IC signals through TLR3. DC maturation basically means that a DC has gotten the signal that it is no longer a sentinel of the immune system but a (terrible terrible over used analogy here) soldier called to arms. In other words, the DC has sensed that some pattern receptor triggering (and generally trouble to the body) invador is present and it undergoes a developmental pathway (maturation) that turns it into a cell that can elicit an immune response (co-stimulatory molecules are increased so that the DC can trigger other cells to activate, antigen presentation on the surface is increased, inflammatory cytokines are released). Poly IC does this through activating the TLR3 signaling pathway.

[Hanna1]

Currently, I am generating Dendritic cells from monocytes by positive
selection using the CD14+ microbeads from milteny and I use as a
complete medium for the culture of monocytes: RPMI FCS 10%, 2Mm
glutamine, 1% P/S supplemented with 1000 U/ ml GM-CSF (Immunotool) and
580 U IL4 (R and D system) and as a dish a Flask. The density of the
cells is 1 million cell/ml DC medium and I replace half of the medium
every 3 days.

While working with fresh PBMC of Buffy coats, I can generate after 6
days immature DC with a yield of 30% of input monocytes. However, while
working with frozen PBMC, the yield of immature DC is too low 10%. The
plastic adherence ii much better, after six days 20% of the input
monocytes are immature DC but they do not fully express CD1a. However
this method could not provide us with a good purity.

Because fresh cells are not always regularly available from myeloma
patients, could you please tell me whether the generation of Dendritic
cells should be made only from fresh PBMC and immature DC are to be
frozen until use or something in my protocol is causing the death of monocytes during culture.

If any one could tell why in some protocl 200 µM ß-mercaptoethanol is used in DC MEDIUM.

thank you

[edelmarierivera]

I have been isolating rat DC from spleen and mesenteric lymph node . In addition, I am trying to isolate rat BMDC. My lab doesn't have and incubator so I have to transfer the petri dishes from my lab to another lab. Since the risk of contamination is higher when you are working with dishes instead flasks.......I am wondering if anybody have been tried to culture them (DC) in flasks???

[Binchen]

The best way I have found to isolate DC from spleen is by first carrying out a colagenase digestion on the spleen, after which Use the MACs system with beads for a specific marker of DC's. This has worked well for me in the past.

Good Luck

Really? Collagenase? I always see activation of my DCs if I digest them with collagenase. Therefore we now use DNase and Liberase, at least when we're interested in maturation markers or T cell stimulation capacity.

I also found that the beads sticking to the DCs alter their appearance in flow cytometry quite markedly so I try to negatively sort them with a cocktail of linage markers for all other stuff.

[Heather T]

Hello! I have been isolating dendritic cells from a mouse spleen and the past three experiements my cells have died by the end of the experiment. This was all of a sudden, the isolation had been working previously. I have narrowed it down to the step where I add FcR blocking reagent, CD11c, wash, resuspend and add to the column. Somewhere in this process they are dying. Does anyone have any tips or suggestions? Thanks so much!

Heather

[luistsg]

I have the same problem iacah # 10,
I've been working with mouse bone-marrow DCs for a while, but all of a sudden all DCs I generate don't have CD11c!! The cells look great under the microscope, and there is a wonderful, tight population when I run the cells (FS vs. SS) with flow cytometry; Any ideas???

please send to: ltsg100@hotmail.com

[emmanuelle]

I have the same problem iacah # 10,
I've been working with mouse bone-marrow DCs for a while, but all of a sudden all DCs I generate don't have CD11c!! The cells look great under the microscope, and there is a wonderful, tight population when I run the cells (FS vs. SS) with flow cytometry; Any ideas???

please send to: ltsg100@hotmail.com

seems I have the same problem
before when we add GM-CSF to bone marrow and one week later, when stained cells with CD11c, almost 80% were CD11c+
but this time, only 20-30% are CD11c positive.
we highly doubt the GM-CSF is not very good and wonder whether we need to make it again

[Skystraw88]

Need protocol of BMDC induce T lymphocytes proliferate.(MLR)