Mice were euthanized by CO2 induced asphyxiation and perfused with 1X Dulbecco's PBS (CellGro) through the left ventricle with drainage mediated by laceration of the inferior vena cava. From individual mice, single cell suspensions of the CNS, brain and spinal cord, were generated by passing the tissue through a 100μm strainer. Mononuclear cells were isolated by Percoll density centrifugation (Sigma-Aldrich). CD4+ T cells were isolated from pooled CNS mononuclear cells using MACS with L3T4 CD4 positive selecting beads (Miltenyi Biotec). Enriched T cells were assessed for antigen specificity and effective 2D affinities using the 2D micropipette adhesion frequency assay using pMHC coated RBC sensors (25 (link), 27 (link), 35 (link)-37 (link)).
Percoll density centrifugation
Manufactured by Merck Group
Sourced in United Kingdom
Percoll density centrifugation is a laboratory technique used for the separation and purification of cells, organelles, and other biological particles based on their density. It involves the use of a colloidal silica suspension that forms a density gradient during centrifugation, allowing the desired particles to be isolated from the complex mixture.
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2 protocols using percoll density centrifugation
1
Isolation and Characterization of CNS-infiltrating CD4+ T cells
2
Isolation of Cellular Components from Healthy Donors
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One millilitre (1mL) of venous whole blood collected from a healthy male donor aged 30 into EDTA Vacutainer tubes was transferred into an eppendorf tube and centrifuged at low speed (600xg, 15 min). Red blood cells (200 µL) were collected from bottom layer. Peripheral blood neutrophils were isolated using Percoll density centrifugation (Sigma-Aldrich, Poole, UK) according to previously described procedure by Matthews et al. (Matthews et al., 2007) . Primary human dermal fibroblasts (neonatal, male foreskin) were obtained from Cascade Biologics (Portland, OR). Human epidermal keratinocytes were isolated from female donors aged 37 to 52 years old according to the previously (Gruber et al., 2012) . This study was approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki principles. Participants gave their written informed consent (EK Nr. 2011/1149).
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