isolating of mouse spleen Dendritic cells by nycodenz - (Mar/12/2007 )

Hi, everyone,

Have anyone had experience in isolating dendritic cells by nycodenz (sigma)? The nycodenz comes as powder. So, how should I make solutions ( with PBS? ) ? Furthermore, what are dendritic cells like after centrifuge? In the unpper, interface or lower supernanant?

Thanks a lot!

Hi,

I use 14,5% Nycodenz solution in RPMI + 10% FCS/ 10mM EDTA / 20mM Hepes. I suspend splenocytes in this medium (without Nycodenz) after isolation (2ml per spleen) and centrifuge it on Nycodenz gradient (530g, 20 min). I use equal volume of Nycondenz solution and suspension of the splenocytes. After the centrifugation dendritic cells are in the interface, between Nycodenz solution and the cell suspension medium. The pellet at the bottom should contain lymphocytes, macrophages, etc.

Let me know if you want the full protocol for the isolation of DCs.

Thank you very much for your reply. Yes, I do need a full protocol for isolation of mouse spleen DC. And how many mice do I need to get a visible interface?

Here's the protocol:

1. To plain RPMI add 1 mg/ml collagenase and 50 ug/ml DNase.
2. Add 3 ml of RPMI/collagense/DNase to appropriate number of wells in a 6-well plate and add a spleen into each well
3. Using mycoinjector inject the media into each spleen and then chop them into small pieces with scissors
4. Incubate at 37C for 45 min
5. Add EDTA to final concentration 10mM to each well
6. Mash the spleens through a filter and transfer the cell suspension into 15 ml tubes and spin down
7. Suspend the pellet in 5 ml of red blood cell lysis buffer and incubate at RT for 10 min
8. Wash the cells with RPMI+10%FCS / 10mM EDTA /20mM Hepes and resuspend in 2 ml/spleen of RPMI+10%FCS / 10mM EDTA / 20mM Hepes
9. Remove the undigested material by filtration and load the cell suspension on equal volume of 14,5% Nycodenz gradient in RPMI/FCS/EDTA/Hepes and centrifuge a 530g, RT, for 20 min
10. Collect the interface and wash with RPMI/FCS/EDTA/Hepes

I usually get good interface from two spleens.

Thanks a lot for the protocol! It's very helpful.