The isolation of mononuclear cells from the tonsil was performed as previously described [28 (link)]. Briefly, mononuclear cells were incubated for 15 min at RT with the lymphocyte-specific antibodies, anti-CD3 (clone 8E6), anti-CD8a (PT36A) (both from Washington State University Monoclonal Antibody Centre, Pullman, WA, USA), anti-CD21 (clone BB6-11C9.6, Southern Biotech, Cambridge Bioscience, Cambridge, UK) and anti-IgM (Clone K52 1C3, Bio-Rad) antibodies in PBS with 2% FBS (FACS buffer). After incubation, the cells were washed twice in a FACS buffer and then incubated with anti-mouse IgG microbeads (Miltenyi Biotec) for 15 min at room temperature. The cells were then washed twice in a FACS buffer and resuspended in a FACS buffer supplemented with 2 mM EDTA (MACS buffer). Cells were then applied to LD columns held in a MidiMACS magnet (Miltenyi Biotec). The flow-through, containing the lymphocyte depleted fraction and the two 1 mL washes with MACS buffer were collected in 15 mL tubes. The cells were washed twice with a FACS buffer and resuspended in 10 mL of RPMI-1640 (Life Technologies) supplemented with 10% FBS (Autogen Bioclear, Calne, UK), (cRPMI), before cell counting by volumetric flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec).
Midimacs magnet
Manufactured by Miltenyi Biotec
Sourced in Germany
The MidiMACS magnet is a laboratory equipment designed for magnetic separation of cells or biomolecules. It provides a stable magnetic field to facilitate the separation and isolation of magnetically labeled samples.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Anti mouse igg microbeads, by Miltenyi Biotec (1 mentions)
Cd133 microbead, by Miltenyi Biotec (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Ls separation column, by Miltenyi Biotec (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Miltenyi ls columns, by Miltenyi Biotec (1 mentions)
Facsaria 1, by BD (1 mentions)
Cd4 cd62l isolation kit, by Miltenyi Biotec (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
Anti mouse cd8 microbeads, by Miltenyi Biotec (1 mentions)
Anti mouse dx5 microbeads, by Miltenyi Biotec (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Cd44 microbeads, by Miltenyi Biotec (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Vacutainer, by BD (1 mentions)
8 protocols using midimacs magnet
1
Isolation of Mononuclear Cells from Tonsil
2
Isolation and Enrichment of CD133+ CSCs
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CD133+ cells were isolated and enriched from SKOV3 cell line using MACS assay according to the manufacturer’s instruction. Briefly, the adherent cells were detached with 2.5% trypsin-EDTA (Gibco, USA), washed and re-suspended in PBS. Fc receptors were blocked with FCR blocking reagent (Miltenyi Biotec., Germany) and then the cells (1 × 107) were incubated with 20 µl of CD133 microbeads (Miltenyi Biotec., Germany) for 20 min on rotator at 4 °C. Subsequently, cells were washed with PBS containing 0.5% bovine serum albumin and enriched by MidiMACS magnet (Miltenyi Biotec., Germany) using LS, MACS manual cell separation columns (Miltenyi Biotec., Germany). CD133+ cells were collected as primary CSCs for further studies.
3
Isolation and Characterization of Lysosomes
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Lysosomes were isolated from MEFs as described previously (Thelen et al., 2017 (link)). Briefly, cells were incubated with superparamagnetic nanoparticles and after a chase period, lysosomes were isolated using Miltenyi LS Separation columns in combination with a MidiMACS Magnet and successful isolation of lysosomes confirmed with an enzymatic assay for ß-Hexosaminidase activity. Isolated lysosomes were lysed using Triton X-100 and the protein concentration determined using the DC protein assay (BioRad).
4
HIV-infected Macrophage Isolation and Characterization
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MDM were infected with HIV NL4.3 BAL-IRES-HSA for 6 days at 37°C with 5% CO2. HIV-1 infection was confirmed by quantitative PCR (qPCR) (66 (link)), p24 ELISA, and surface expression of virus-encoded murine heat-stable antigen (HSA) by flow cytometry (24 (link)). HSA-expressing MDM were isolated by positive selection using Miltenyi LS columns in combination with the MidiMACS magnet (Miltenyi Biotech). The HSA sorting protocol was optimized by the laboratory of Michel Tremblay, as described previously (24 (link), 25 (link)). Purity of the HSA+ and HSA− fractions was assessed by flow cytometry.
5
Isolation of Lymphocyte Subsets from Mouse Spleen
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Different lymphocyte subsets were purified from splenic mononuclear cells isolated from Balb/c mice (Charles River Laboratories, Wilmington, MA, USA). If necessary, further isolation was performed by magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) or by FACS (FACSAria I, BD Biosciences, San Jose, USA).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 μm wire mesh. DX5+ cells were purified using anti-mouse-DX5+ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). DX5+ cells were collected in the positive fraction. DX5+ splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further DX5+NKT cell isolation by FACS sorting. CD4+CD62Lhigh and CD4+CD62Llow cells were purified using the CD4+CD62L+ Isolation Kit (Miltenyi Biotec) and CD8+ cells by using anti-mouse-CD8+ MicroBeads (Miltenyi Biotec).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 μm wire mesh. DX5+ cells were purified using anti-mouse-DX5+ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). DX5+ cells were collected in the positive fraction. DX5+ splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further DX5+NKT cell isolation by FACS sorting. CD4+CD62Lhigh and CD4+CD62Llow cells were purified using the CD4+CD62L+ Isolation Kit (Miltenyi Biotec) and CD8+ cells by using anti-mouse-CD8+ MicroBeads (Miltenyi Biotec).
6
Isolation and Enrichment of CD44+ CSCs
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Magnetic-activated cell sorting (MACS) technique was used to isolate and enrich CD44+ cells. In brief, the cells were harvested using 0.25% trypsin-EDTA (Gibco, USA). The detached cells were washed and re-suspended in PBS buffer. In the following, 1 × 107 cells were labeled with 20 μl of CD44 microbeads (Miltenyi Biotec., Germany) for 20 min at 4 °C on a rotator. Next, the cells were washed with PBS containing FBS (0.5%) and enriched with LS column (Miltenyi Biotec., Germany) attached to a Midi-MACS magnet (Miltenyi Biotec., Germany). At the end of separation, CD44+ cells were harvested as primary CSCs for subsequent analysis [11 ].
7
Lanthanide-Doped SPIO Nanoparticle Synthesis
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Dextran coated, lanthanide doped, SPIO nanoparticles were prepared though the coprecipitation of ferrous, ferric, and lanthanide ions in the presence of dextran as described previously53 (link). After the reaction, the Ln-SPIO was purified by diafiltration across a 100 kDa membrane and was 0.2 μm filtered to remove any oversized material. Finally, to ensure complete purification of the Ln-SPIO from excess salt, lanthanide ions, and FITC, the nanoparticles were magnetically purified on MACS LS columns using a MidiMACS magnet (Miltenyi Biotec, Auburn, CA, USA). Control particles, i.e. those not labeled with targeting ligands, were reacted at a 1:10 molar ratio of SPIO:FITC in pH 9 sodium bicarbonate buffer. The labeled particles were purified on a PD10 gel filtration column in PBS to yield approximately 3 dye molecules per particle.
8
Isolation and Analysis of CD14+ Monocytes
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Mononuclear Cells (PBMCs) and Sorting of Cluster of Differentiation (CD)-14 1 and CD14 2 PBMCs Blood samples were stored at room temperature and processed within 24 h after sampling. Peripheral blood was collected in EDTA tubes (Vacutainer, BD Biosciences) for whole blood analysis, and PBMCs were isolated from heparinized blood as previously described [20] (link). CD14 ? monocytes were isolated from PBMCs using CD14 MicroBeads (1:10), LS columns, and a MidiMACS magnet according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) [21] . The throughputs of the CD14 MACS sort depleted of CD14 ? monocytes served as CD14 -fractions of PBMCs. Since we hypothesized that T. whipplei more reliably persist in CD14 ? monocytes, the fraction depleted of CD14 ? cells was intended to serve as a kind of negative control with lower detection rates. CD14 ? purity of the CD14 ? fractions was estimated using flow cytometry with a CD14 (61D3) antibody from eBiocience 1.59 ± 2.04%; p = 0.003). CD14 -fractions were analyzed for only five patients with WD and revealed a contamination of 8.56 ± 4.64% of CD14 low and 0.70 ± 0.44% of CD14 high monocytes.
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