Cells clots forming during preparation mouse splenocytes - (Jun/21/2009 )
Hi,I am working on immunity of mouse infected with malaria,I have a trouble when preparing splenocyes.The splenocytes were clotted after digested with Collagenase D and Dnase I,then I can do nothing because of this big problem.By the way,I tried to dose not add EDTA when neutralizing the enzyme reaction,clots did not appear,but the most of the splenocytes were died.The mice were sacrificed when 15 days infected with Pb malaria.In addition,the normal(non-infected with malaria)mice have no this problem. I used this protocal to prepare splenocytes.
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.
look forward to hearing from you.
Is there anyone who is working on malaria infection and immunity?
Is it necessary to stop the collagenase D digestion with EDTA for preparing splenocytes?
I tried several times,it is no necessary to stpo using EDTA.