LNSCs were obtained as previously described (Link et al., 2007 (link)). In brief, total skin or skin-draining LNs from individual or 10–15 pooled mice were cut into small pieces and digested in RPMI containing 1 mg/ml Collagenase IV (Worthington Biochemical Corporation), 40 µg/ml DNase I (Roche), and 2% FBS. Undigested cells were further digested with 1 mg/ml Collagenase d , and 40 µg/ml DNase I (Roche). The reaction was stopped by addition of 5 mM EDTA and 10% BSA. Samples were further disaggregated through a 70-µm cell strainer and blocked with anti-CD16/32 antibody. Single cell suspensions were negatively selected using CD45 microbeads and magnetic bead column separation (Miltenyi Biotec).
Magnetic bead column separation
Manufactured by Miltenyi Biotec
The magnetic bead column separation is a laboratory equipment designed for the isolation and purification of target cells, proteins, or other biomolecules from complex samples. It utilizes the principle of magnetic separation to effectively capture and separate the desired components from the sample mixture.
Automatically generated - may contain errors
3 protocols using magnetic bead column separation
1
Isolation of Skin-Draining Lymph Node Cells
2
Isolation of Mouse Aortic and Lymphatic Endothelial Cells
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Thoracic-abdominal aortas from Panx1delApoe−/− or Panx1
f/flApoe−/− mice were immerged in 100–150 μl PBS on a silicon support, longitudinally opened and pinned. The endothelial side was then delicately scraped with a scalpel blade. The samples were centrifuged 5 min at 1350 rpm and immediately processed for RNA extraction.
LN lymphatic ECs were obtained from skin LN isolated from 8–10 mice and digested in RPMI containing 1 mg/ml Collagenase IV (Worthington Biochemical Corporation), 40 µg/ml DNase I (Roche), and 2% fetal bovine serum gently mixing the samples every 10 min for a total incubation of 30 min. Undigested cells were further digested with 1 mg/ml Collagenase D, and 40 µg/ml DNase I (Roche) for not more than 20 min. The enzymatic reaction was stopped by addition of FACS buffer. Single cell suspensions were negatively selected using CD45 microbeads and magnetic bead column separation (Miltenyi Biotec).
f/flApoe−/− mice were immerged in 100–150 μl PBS on a silicon support, longitudinally opened and pinned. The endothelial side was then delicately scraped with a scalpel blade. The samples were centrifuged 5 min at 1350 rpm and immediately processed for RNA extraction.
LN lymphatic ECs were obtained from skin LN isolated from 8–10 mice and digested in RPMI containing 1 mg/ml Collagenase IV (Worthington Biochemical Corporation), 40 µg/ml DNase I (Roche), and 2% fetal bovine serum gently mixing the samples every 10 min for a total incubation of 30 min. Undigested cells were further digested with 1 mg/ml Collagenase D, and 40 µg/ml DNase I (Roche) for not more than 20 min. The enzymatic reaction was stopped by addition of FACS buffer. Single cell suspensions were negatively selected using CD45 microbeads and magnetic bead column separation (Miltenyi Biotec).
3
Differentiation of Dendritic Cells and Macrophages from Bone Marrow
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DCs were generated by culturing BM cells for 7–10 d in the presence of 20 ng/ml GMCSF in RPMI supplemented with 10% heat-inactivated fetal bovine serum, 50 mM 2-mercaptoethanol, 100 mM sodium pyruvate, and 100 µM penicillin/streptomycin at 37°C, 5% CO2. For some experiments, DCs were treated with 1 µg/ml LPS (Enzo Life Sciences) for the last 16 h of culture. For macrophage differentiation, BM cells were cultured for 7 d in DMEM containing 4.5 glucose, supplemented with 20% FBS, 30% L929 supernatant, and 1% penicillin/streptomycin. B cells were purified from the spleen of naive WT mice using the CD19 depletion kit and magnetic bead column separation (Miltenyi Biotec).
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