The mechanical dissociation of the brain cortex tissue was performed using the Adult Brain Dissociation Kit (Miltenyi Biotec, Germany) and Gentle MACS dissociator (Miltenyi Biotec) according to the manufacturer’s protocols. Endothelial and microglial cells were separated from the remaining cells using magnetic cell sorting. For endothelial cell separation, the cells from the whole brain cortex (n = 4, each group) were resuspended with CD45 microbeads (Miltenyi Biotec) and passed through an MS column. The flow-through containing CD45-negative unlabeled cells was collected and centrifuged (300 × g, 10 min). After removal of the supernatant, the cells were incubated with CD31 microbeads (Miltenyi Biotec) and then passed through an MS column. The CD31-positive cells that remained in the column were washed out and collected by firmly pushing the plunger into the column. CD45-negative cells and CD31-positive cells were isolated using the above procedure. For microglial cell separation, the cells were resuspended with CD11 microbeads (Miltenyi Biotec), and then the CD11-positive cells that remained in the MS column were washed out and collected by firmly pushing the plunger into the column. Microglial cells were counted using a hemocytometer.
Cd11 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD11 microbeads are magnetic beads coated with antibodies specific for the CD11 antigen. They can be used to isolate cells expressing the CD11 marker from complex cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Cd31 microbead, by Miltenyi Biotec (1 mentions)
Adult brain dissociation kit, by Miltenyi Biotec (1 mentions)
Cd45 microbeads, by Miltenyi Biotec (1 mentions)
Nf κb p65 elisa kit, by Enzo Life Sciences (1 mentions)
Model 550, by Bio-Rad (1 mentions)
Mouse il 10 quantikine elisa kit, by R&D Systems (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Mouse il 23 quantikine elisa kit, by R&D Systems (1 mentions)
2 protocols using cd11 microbeads
1
Brain Cortex Cell Isolation
2
CD11c+ DCs Cytokine Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
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CD11c+ DCs from lymph nodes were selected with anti-CD11c beads according to the protocol of CD11 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured in a Roswell Park Memorial Institute (RPMI) medium 1640 with 10% fetal bovine serum. DCs (1.0 × 105 cells per 200 μL well) were stimulated with 1 μg/mL of R848 (InvivoGen, San Diego, CA, USA) for 24 h and the total RNA was then isolated using Trizol (Invitrogen, Waltham, MA, USA). Then, the mRNA expression levels were determined as mentioned above. The supernatants were also harvested and the IL-23 and IL-10 protein levels were quantified using a mouse IL-23 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and a mouse IL-10 Quantikine ELISA Kit (R&D Systems), according to the manufacturer’s instructions. In some experiments, the DCs were stimulated with 1 μg/mL of R848 for 15 min or 1 h and the phosphorylated NF-κB p65 expression in DCs was analyzed with a NFκB p65 ELISA kit (Enzo Life Sciences, Tokyo, Japan) according to the manufacturer’s instructions. These assays employed the quantitative sandwich enzyme immunoassay technique. Optical densities were measured at 450 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
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