Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE

Abstract

ObjectiveTo determine the capacity, effectiveness, efficiency, and reliability of select tissue dissociation methods to isolate mononuclear cells from the CNS of mice with experimental autoimmune encephalomyelitis (EAE).MethodsAs part of an assay qualification, we tested the isolation method Percoll PLUS vs a commercially available enzymatic Neural Tissue Dissociation Kit (Kit), and the enzymes accutase and papain in C57BL/6 mice with active EAE. In a stepwise approach, we applied the following 4 criteria to each dissociation method: (1) mononuclear cell viability post-processing was required to be ≥80% per brain or spinal cord sample, (2) absolute live mononuclear cell numbers was required to be ≥5 × 105 per brain or spinal cord sample of mice with clinical EAE, (3) test-retest reliability had to be verified, and (4) the absolute mononuclear cell numbers in brain and spinal cord had to correlate with the EAE disease course.ResultsEnzymatic dissociations allowed for greatly increased cell yield and specifically allowed for downstream assays from individual brains and spinal cords in C57BL/6 mice with EAE. All enzymatic dissociations provided a more efficient and effective method for isolating mononuclear cells from brains and spinal cord. Only the Kit assay provided a significant correlation between absolute mononuclear cell numbers in the spinal cord and EAE disease severity.ConclusionsEnzymatic dissociation of CNS tissue of C57BL/6 mice with active EAE with the Kit should be the standard method. The identification of optimized CNS dissociation methods in EAE has the potential to identify cellular events that are pertinent to MS pathogenesis.

α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model

“…To induce active EAE, experimental mice were immunized subcutaneously with myelin MOG p35-55 (200 μg/100 μL/mouse), emulsified in an equal volume of complete Freund adjuvant containing 4 mg/mL H37Ra Mycobacterium tuberculosis (Difco, BD, Franklin Lakes, NJ) in each flank as described. 13…”

CD11c+CD88+CD317+myeloid cells are critical mediators of persistent CNS autoimmunity

“…Cells from lymph nodes and spleen were isolated by pressing through a 70 µm nylon mesh cell retainer as described [8][9][10] . Cells were treated with red blood cell (RBC) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), washed twice with cold phosphate buffered saline (PBS), and re-suspended in PBS for counting by hemocytometer.…”

“…Mice were anesthetized with 250 mg/kg i.p. injection of tribromoethanol (Avertin, Sigma Aldrich, St Louis, MO) solution and perfused with PBS through the left ventricle as described 8 . Brains were dissected from the skull and spinal cords were flushed from the spinal column with PBS.…”

TLR3 agonism re‐establishes CNS immune competence during α4‐integrin deficiency

“…As previously described, 26 brains and spinal cords were first finely minced using a sterile scalpel, washed with cold PBS, then processed based on the specific enzymes used. The commercially available Neural Tissue Dissociation Kit (P) (Kit) was used following the manufacturer's protocol (Neural Tissue Dissociation Kit (P), Miltenyi Biotec, San Diego, CA, USA).…”