I'm trying to isolate viable single cell suspension from brains harvested from 2 month old mice/rat. I'm specifically looking for enzymatic methods of dissociation. I understand pappain works better than collagenase+hyaluronidase mix. But wanted to know if anyone has an effective working method. Any help would be appreciated!
If the vibration isolation method is good to isolate neurons from young rat brain (P9-P20, but not applicable in adult brain and also is not applicable for astrocyte isolation, the enzymatic method(s) are used in our labs. We use papain (20-40 Units/mL) in PBS for 20-40 minutes at 37*C tissue incubation;
and then after wash in L15 (or MEM, or ECSF) use DNase from bovine pancreas (play with concentration in ACSF), but for very short time (1-20 seconds, with following wash in ACSF or in MEM. Better to use small tissue fragments for enzyme deep permeability. Please, also check Min Zhou papers for his method. Papain emulsion is good from Boehringer or SIgma-Oldrich, or Miltenyi Biotec - miltenyibiotec.com. DNase from Sigma-Oldrich.
If the vibration isolation method is good to isolate neurons from young rat brain (P9-P20, but not applicable in adult brain and also is not applicable for astrocyte isolation, the enzymatic method(s) are used in our labs. We use papain (20-40 Units/mL) in PBS for 20-40 minutes at 37*C tissue incubation;
and then after wash in L15 (or MEM, or ECSF) use DNase from bovine pancreas (play with concentration in ACSF), but for very short time (1-20 seconds, with following wash in ACSF or in MEM. Better to use small tissue fragments for enzyme deep permeability. Please, also check Min Zhou papers for his method. Papain emulsion is good from Boehringer or SIgma-Oldrich, or Miltenyi Biotec - miltenyibiotec.com. DNase from Sigma-Oldrich.
Worthington is an enzyme manufacturer that has very good protocols and better prices than distributors.
Would it be an option to isolate single cells from brain tissue by laser microdissection?
http://www.molecular-machines.com/single_cell_sorting_products/_mmi_cellcut_-_laser_microdissection/technology_27
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