Splenocytes were isolated from C57BL/6 or BALB/c mice. The granulocytes and RBCs were removed by Ficoll (Sigma-Aldrich) density gradient centrifugation. After the depletion of CD3ɛ+ and DX5+ cells from the cell population using anti-mouse CD3ɛ and anti-mouse DX5 microbeads, the CD11b+ cells and B220+ cells were purified using anti-mouse CD11b and anti-mouse B220 microbeads (all from Miltenyi Biotec). Isolated B cells and monocytes (1:1 ratio mixture) were transduced with the indicated adenoviral vector in a 9-min, 2,800-r.p.m. centrifugation step at room temperature, and the cells were subsequently incubated for an additional 18 h (for the B/Mo/αGC preparation this step was skipped). αGC (KRN7000, Enzo Life Science, Japan) was loaded into the prepared B cells and monocytes for NKT activation based on our previous studies20 (link)21 (link)22 (link)51 (link).
Krn7000
Manufactured by Enzo Life Sciences
Sourced in Japan
KRN7000 is a synthetic glycolipid compound. It is used as a research tool in biochemical and cellular studies.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll, by Merck Group (1 mentions)
Anti mouse dx5 microbeads, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Rhil 2, by R&D Systems (1 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Rhil 7, by R&D Systems (1 mentions)
Rhil 15, by R&D Systems (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Easysep human cd8 t cell enrichment kit, by STEMCELL (1 mentions)
3 protocols using krn7000
1
Purification and Activation of B Cells and Monocytes
2
Staining and Analysis of iNKT Cells
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KRN7000 (Enzo Life Sciences) was prepared and stored as described55 (link). Animals were injected intravenously with 5 μg KRN7000 and organs were isolated 2 hours later. Surface staining was performed as described above. Cells were fixed and permeabilized following kit instructions (Cytofix/Cytoperm Kit, BD Pharmingen), then stained intracellularly for IFN-γ (XMG1.2) and IL-4 (11B11). iNKT cells were identified by expression of TCR-β and NK1.1 due to down-regulation of TCR expression.
3
Isolation and Expansion of Human T Cell Subsets
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Human peripheral blood mononuclear cells (PBMCs), obtained from Shanghai Public Health Clinical Center, were sorted into CD4+ and CD8+ T cells by EasySep Human CD4+ T Cell Enrichment Kit (STEMCELL #19052) and EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL #19053), respectively. After activation by anti-CD3/CD28 immunobeads in T-cell growth medium (TCM) for 24–36 hours, the purified T cells were transduced by lentiviruses in a novonectin (Novoprotein)-coated 48-well flat plate by spin infection. The next day, the cells were fed with fresh TCM. The immunobeads were removed 6–7 days after activation, and cells were expanded followed by 3-hour rest before being assayed. iNKT cell expansion iNKT cells were expanded from human PBMCs in X-VIVO-15 Medium (Lonza) containing 100 ng/mL KRN7000 (Enzo Life Science), 100 U/mL rhIL-2 (R & D Systems), 20 ng/mL rhIL-7 (R & D Systems) and 20 ng/mL rhIL-15 (R & D Systems) for 20 days as previously described.23 24 (link)
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