Normally I isolate DCs from lymph nodes using Collagenase D and DNase I. However I know I plan to try isolation of DCs from the lamina propria and some protocols suggest Collagenase VIII instead. Did someone compare the yield of DCs from the gut using different types of collagenases?
Chantal,
Both Collagenase D and Collagenase VIII should work. If you have a good DC recovery from Collagenese D - keep use it, otherwise try to use Collagenese VIII. Incubation time and concentration for Collagenese VII will be different.
Chantal,
Normally, I use 500 U/ml Collagenase IV to digest lamina propria for 1 hour @ 37 oC. You can see a very good DC population after digest. Good luck.
I agree with what has been said above; however, you may also try Collagenase XI.
Zhi-Chun Ding, could you give me your exact protocol? Do you use Percoll afterwards? My mail is [email protected]
Thanks to all for your help! Up to now I got the best results with MIltenyi lamina propria dissociation kit (but very few cells) and with Collagenase VIII (but I have many dead cells and tissue cells ... probably percoll would be good)
Chantal,
1. chop the tissue into very small pieces first.
2. add 0.5 ml 500 U/ml collagenase (worthington) in RPMI containing 1% FBS.
3. Digest samples in incubator at 37 oC for 1 hours.
4. Pipet samples up and down with 1 ml tip, and pass samples through a 70 um strainer.
5. spin cells and resuspend cells in PBS for FACS staining and analysis.
I don't use Percoll after digestion. Good luck.
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