23 replies to this topic

[Dirk Benke]

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

[Darren]

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.

[Douglas vasey]

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

[guineth]

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

[homa]

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

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

[Nexus]

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.

Edited by Nexus, 04 October 2004 - 08:56 AM.

[immune platelet]

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.

[raghvendra]

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 ? )

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 ? )

[DendriticDude]

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

[iacah]

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???

[dmcallister]

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

[klonegenie]

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........

[amaterasu]

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

[Davi]

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

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

[Shankar]

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