1.Spleen was placed in 4ml enzyne cocktail containing 100U /ml collagenase D(Roche) and 20ug/ml DNase I.Hold spleen with forceps and push onto 22-G needle while slowly injecting 1ml enzyne solution.
2.Put the cell solutions to ice.Tease the spleen apart into small fragments using forceps.
3.Add 4 ml 400U/ml collagenase to dish with leftover fragments and incubate at 37oC for 60 minutes.All the enzyene is dissolved in HBSS with Ca+ and Mg+.
4.The cell suspension was transferred to 50ml tube containing 15ml of 2mM EDTA and HBSS without Ca+ and Mg+ to stop the enzyme reaction.
Edited by Davi, 21 June 2009 - 08:38 PM.