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Posted by: Dirk Benke Mar 13 2002, 10:18 AM

This is my first post in this Forum.
Therefore I would like to know, if anyone can help me with a protocol of DC (Dendritic-Cell) Isolation from spleen or even of other organs.

Thank you

Posted by: Darren Apr 8 2002, 11:45 AM

have you tried magnetic bead(Dynabead) separation using say a specific marker for DC's eg.,CD11b/c,CD 34.
or you could remove murine femur and flush out the bone marrow and try pushing the stem cells into a DC lineage using IL-4 and GM-CSF.
Darren E.

Posted by: Douglas vasey Apr 20 2004, 07:35 AM

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

Posted by: guineth Jun 1 2004, 12:16 PM

My boss asked me to begin to work with IMMATURE dendritic cells but I never isolated dentritic cells and I dont know anything about this technic (usually I am doing proteomics, I did not touch a mouse of my whole life!!!). Do you have a protocol to isolate dc from mouse femur bone marrow , and how to grow them? I have a very poor background in immunology so anything you can tell me will be helpfull.

Thank you very much

Posted by: homa Jul 26 2004, 12:20 AM

For this purpose U can isolate Bone marrow cells from femur bone of mouse with pushing and then culture them.
1- kill the mouse and isolate femur
2- Cut the haed of bone and use a insulin syringe to push cells in a pettry dish
3- Centrifuge in 300 G for 10 min
4- Then lysis the RBC with lysis buffer
5- Culture the cells with GM-CSF(20ng/mm) plus IL-4 (1000u/mm)
6- change meddium with new one every 3 days
7- At 7th day U have immature DC and if add TNF-a or maturation factore U will have Mature DC

Have a nice time

Posted by: Nexus Oct 4 2004, 07:31 AM

Hello folks

I'm bringing this discussion back on the spotlight. I need to reach a very high purity of immature and mature DC 's for RNA isolation in order to analyse the presence of some receptors on DC's.

Does anyone have any ideea what markers can be used for at least 99% concentration. I suggested a Cd11c + B220- and MHC II + sort but I'm not sure that will grant me a very high purity.


Thank you very much for any suggestion.

Posted by: immune platelet Oct 12 2004, 10:00 AM

Miltenyi has a very nice isolation method. It's simple, label your DC with CD11c-FITC or CD11c-PE and isolate them with anti-FITC or anti-PE conjugated beads. Works very well (>90% purity) for spleen and lymph node. We are still working out the bugs with other tissues and thats due to the debris from organs like lung and liver.

Posted by: raghvendra Oct 31 2004, 12:45 AM

how many workers on dendritic cell believe that thsese are nonadherent cells only , i think in presence of GMCSF and IL4 many cell remains attached which shows almost all charecetrs of dendritic cells and not exclusively the morphology is criteria for calling them DCs ; immature DCs never shows dendritic morphology only full blown DCs shows dendritic structure and blunt dendrities ;
i have tried to generate DCs from human , rat, mice hamester, guinea pig and rabbit , infact different species DCs donot response same for their differentiation toward immature DCs, many of guyinea pig PBMC even donot survive in the same titre of GMCSF which allows mice and other species PBMCs to direct toward DCs , i want the open answer for this do u all believe the same ; u can try also,,,
only beauty og bone marrow DCs is that it gives an synchronised homogeneous DCs for experiments becs DCs isolated from animals direct with markers like CD11c and OX-62 can not differentiate between immature an dmature stage which is the crucial most aspect of studying now a days ( am i right ? )

Posted by: DendriticDude Nov 25 2004, 09:43 PM

I can email a detailed (5 page) protocol which has been the basis for experiements by several major DC characteristion groups. Please email me directly if you still require this.

simpsoc@svhm.org.au

Posted by: iacah Jan 13 2005, 11:01 AM

I've been working with mouse bone-marrow DCs for a long time, 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; however, it's hard for me to justify saying that these are DCs when there is no CD11c showing up on them. Any ideas???

Posted by: dmcallister Jan 31 2005, 12:21 PM

Does any one have a good idea of how many DCs should be recovered from a normal mouse spleen?

Posted by: klonegenie Feb 18 2005, 03:25 PM

for isolation of DC from mouse spleen.the best protocol is described in RM steinman's pioneering paper of 70's in Journal of experimental medicine. u can modify it according to ur need........

Posted by: amaterasu Mar 23 2005, 04:00 AM

preparation of DC from mouse bone marrow (BMDC)

kill mouse (I've always used BALB/C, but the protocol is accurate also for other stems), e.g. by cervical dislocation
cleanly excise both femurs and remove excess tissue
cut off both ends of each femur and flush out bone marrow with a 27G needle (attached to a syringe with DC complete medium = DCCM)
resuspend gently to dissolve cell aggregates
pellet suspension for 5 min at 1300 - 1500 rpm and resuspend then in 1 ml DCCM
perform erythrocyte lysis with dH2O (3 ml for exactly 15 sec) and "stop" lysis by adding 3 ml 0.3M NaCl (i/c)
centrifuge again and resuspend in suitable vol DCCM
seed into 10 cm diameter bacteriological grade dishes (in 10 ml) - I recommend Falcon-1001 or Greiner)
keep at 37°C, 7% CO2 and feed every 3 days by adding 10 ml fresh DCCM or - dep on the cell density - split on day 6 into 2 dishes (10 ml each + 10 ml fresh medium)
at day 10, app. 10e7 immature DC can be harvested by pipetting (supsension cells)

note: cells will mature automatically by prolonged culture periods (use between day 10 and max. 14 for experiments)

DCCM:
RPMI-1640
5% FCS
2 mM L-glutamine
1% pen/strep (sigma)
200 µM ß-mercaptoethanol
10% GM-CSF conditioned supernatant (produced by GM-CSF-secreting cell line) OR 1-2 ng/ml rGM-CSF

Posted by: Davi Jun 21 2009, 09:23 PM

Some papers say DC should comprise 7% of the total splenocytes using collagenase D digestion method.Right?

Posted by: Shankar Jun 22 2009, 07:38 AM

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

Posted by: Astarte Biologics Jul 13 2009, 01:09 PM

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

Posted by: miBunny Jul 14 2009, 06:38 PM

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.

Posted by: Hanna1 Aug 27 2009, 07:45 AM

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

Posted by: edelmarierivera Sep 8 2009, 07:21 AM

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???

Posted by: Binchen Sep 30 2009, 06:38 PM

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.