Primary rest NK cells were separated from peripheral blood mononuclear cells (50mL; from healthy donors). The mononuclear cells were purified using Ficoll Paque density gradient centrifugation with 15mL falcons (BD, CA). Cell sorting from peripheral blood mononuclear cell was performed using the antibody-conjugated magnetic beads (Miltenyi Biotech, Gladbach, Germany) by negative selection kits, according to the manufacturer’s instructions. Twenty million mononuclear cells were used as the input count of cells for all the bead sorting. Mononuclear cells underwent 2 rounds of sorting through CD3 and thereafter CD14 beads (2mL beads/1 million cells). Flow cytometry was used to assess enrichment populations (more than 95% purity). The process was followed by expansion and activation in XVIVO-20 medium (Lonza, Barcelona, Spain) supplemented with 10% fetal bovine serum (Gibco, UK), 500IU/mL IL-2 (Promokine, Germany), 10ng/mL IL-12 (Promokine), 100ng/mL Galactosyl ceramide, 50ng/mL IL-15 (Promokine), 1μM Valproic acid, and 10ng/mL IL-21 (Promokine) for about 5 days.
Antibody conjugated magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Antibody-conjugated magnetic beads are small, uniform particles coated with antibodies. They serve as a tool for isolating and purifying specific target cells or molecules from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by BD (1 mentions)
X vivo 20 medium, by Lonza (1 mentions)
Taqman system, by Thermo Fisher Scientific (1 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Lymphoprep, by Abbott (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
5 protocols using antibody conjugated magnetic beads
1
Isolation and Expansion of Primary rNK Cells
2
Blood Plasma Collection and Cell Isolation
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Blood was collected in EDTA tubes and processed within 3 hrs. Plasma was separated and stored at −80oC for ELISA and bead-based multiplex assays. PBMCs were separated by Ficoll-gradient centrifugation followed by purification of CD14+ monocytes by negative selection with antibody-conjugated magnetic beads according to the manufacturer's instructions (Miltenyi Biotech).
3
Detecting EBV-Infected Cell Populations
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Infected cells were detected and isolated as described previously [37 (link)]. Briefly, the PBMCs obtained from patients were isolated by density gradient centrifugation using Separate-L (Muto Pure Chemical, Tokyo, Japan) and were sorted into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany; 130-050-301, 130-045-101, 130-045-201, 130-090-875). After that, the EBV DNA levels in each fraction were evaluated by real-time PCR using the TaqMan System (Applied Biosystems, Foster City, CA) [38 (link)].
4
Isolation of EBV-Infected Cell Subsets
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The EBV-infected cells were isolated as described previously (Onozawa et al., 2018 (link)). In brief, the peripheral blood mononuclear cells (PBMCs) from patients were isolated by density gradient centrifugation using Lymphoprep™ (Abbott Diagnostics Technologies AS, Oslo, Norway) and sorted into CD4-, CD8-, or CD56-positive fractions by using antibody-conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The primary monocytes were obtained by negative selection assay using Pan Monocyte Isolation Kit (Miltenyi Biotec).
5
Monocyte Gene Expression Profiling
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Monocytes were purified for gene expression analysis at weeks 0, 6, 12, and 18 (washout). Blood was collected in EDTA tubes and peripheral blood mononuclear cells (PBMC) separated by Ficoll-gradient centrifugation. CD14+ monocytes were then isolated by negative selection with antibody-conjugated magnetic beads according to the manufacturer's instructions (Miltenyi Biotech), suspended in RNAlater (Ambion), and frozen at −80°C until RNA extraction. FACS analysis was done on each isolation and based on CD14+ staining, mean monocyte purity was 93% (range: 92%-94%).
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