PMC:4136538 / 8722-11591
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4136538","sourcedb":"PMC","sourceid":"4136538","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4136538","text":"As described previously [4], following transcardiac perfusion with phosphate buffered saline (PBS), mononuclear cells for flow cytometric analysis were prepared from an individual spleen, pooled lumbar lymph nodes (LNs) (5 mice), or pooled mouse lumbar spinal cord tissue (10 mice) by discontinuous Percoll (Amersham, Piscataway, NJ) gradients, and the total mononuclear cell number was determined using a hemacytometer with trypan blue (Sigma, St Louis, MO) prior to intracellular flow cytometric analysis. Due to the limited number of infiltrating cells available for analysis following the collection procedure (even with 10 mice pooled together) and the fact that the vast majority of infiltrating CD4+ T lymphocytes were observed in the ipsilateral side of dorsal horn area [4], lumbar spinal cord tissues were not separated as ipsilateral vs. contralateral in these experiments. In general, the intracellular staining was performed as follows. Briefly, following the final PBS wash, mononuclear cells were resuspended in staining buffer (2% fetal bovine serum (FBS)/0.09% NaN3/PBS) containing anti-mouse-CD16/CD32 monoclonal antibody (mAb) (clone 2.4G2; 1 µg/50µl/tube) and incubated on ice for 30 minutes. A combination of three fluorescent mAbs (Table 1) was added to each tube for surface staining (30 minutes on ice). After two PBS washes, the cells were fixed with 1% formaldehyde/PBS at room temperature for 20 minutes, washed with PBS again, and then permeabilized with a permeabilization buffer (0.5% saponin (Sigma)/2% FBS/0.09% NaN3/PBS) at room temperature for 10 minutes. Cells were then blocked with 10% isotype-matched serum (either mouse or rat serum) (in 0.5% saponin/PBS) on ice for 30 minutes. One selected fluorescent mAb (Table 1) was added to each tube for intracellular staining (30 minutes on ice). After being washed with permealization buffer, cells were resuspended in 1% formaldehyde/PBS and stored at 4°C until flow cytometric analysis. All samples were analyzed with an Accuri C6 flow cytometer (BD Biosciences-Accuri, Ann Arbor, MI) and FlowJo analysis software (Tree Star, Inc., Ashland, OR). For spleen and LN samples, a total of 20,000 events were collected and analyzed. For spinal cord mononuclear cells, due to the limited numbers of cells obtained, all events in each sample were analyzed. Non-stained cells, proper isotype controls, and surface-stained only controls were included as controls. For intracellular cytokine staining IFN-γ, IL-4, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF)), 0.1% brefeldin A (Sigma)/PBS was used in place of PBS from tissue harvesting up to the fixation step. All experiments were repeated several times in order to collect sufficient data for statistical analyses (i.e., when pooled samples were used, each pooled sample was considered as n=1).","tracks":[{"project":"2_test","denotations":[{"id":"25143871-18196515-63946020","span":{"begin":25,"end":26},"obj":"18196515"},{"id":"25143871-18196515-63946020","span":{"begin":25,"end":26},"obj":"18196515"},{"id":"25143871-18196515-63946021","span":{"begin":780,"end":781},"obj":"18196515"},{"id":"25143871-18196515-63946021","span":{"begin":780,"end":781},"obj":"18196515"},{"id":"T93657","span":{"begin":25,"end":26},"obj":"18196515"},{"id":"T86722","span":{"begin":780,"end":781},"obj":"18196515"},{"id":"T19054","span":{"begin":780,"end":781},"obj":"18196515"},{"id":"T25080","span":{"begin":25,"end":26},"obj":"18196515"}],"attributes":[{"subj":"25143871-18196515-63946020","pred":"source","obj":"2_test"},{"subj":"25143871-18196515-63946020","pred":"source","obj":"2_test"},{"subj":"25143871-18196515-63946021","pred":"source","obj":"2_test"},{"subj":"25143871-18196515-63946021","pred":"source","obj":"2_test"},{"subj":"T93657","pred":"source","obj":"2_test"},{"subj":"T86722","pred":"source","obj":"2_test"},{"subj":"T19054","pred":"source","obj":"2_test"},{"subj":"T25080","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ecec","default":true}]}]}}