Neutrophils were isolated from whole blood using Mono-Poly resolving medium (DS Pharma Biomedical), as described previously (71 (link)). We seeded neutrophils in an 8-well chamber slide (Lab-Tek) in 400 μL of serum-free medium, RPMI-1640, at a density of 105 cells/well. PBMCs were isolated from whole blood using Ficoll-Paque PLUS (GE Healthcare). CD4, CD8, HLA-DR, and CD14 positive cells were isolated from PBMCs using CD4, CD8, HLA-DR, and Monocyte Isolation Kits (Miltenyi Biotec) per the manufacturer’s protocol. NHEK was purchased from Kurabo and cultured on EpiLife medium supplemented with insulin (10 mg/mL), rhEGF (epidermal growth factor, 0.1 ng/mL), hydrocortisone (0.5 mg/mL), gentamicin (50 mg/mL), amphotericin B (50 ng/ml), and bovine pituitary extract (0.4% v/v); EpiLife-KG2 medium, all from Kurabo) in a humidified atmosphere with 5% CO2 at 37°C. Monocytes in blister fluids were isolated from whole blister fluids using a Monocyte Isolation Kit (Miltenyi Biotec) per the manufacturer’s protocol.
Monocyte isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, United Kingdom
The Monocyte Isolation Kit is a laboratory product designed to isolate monocytes from peripheral blood or other biological samples. It utilizes a procedure that allows for the separation and enrichment of monocytes from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Nk cell isolation kit, by Miltenyi Biotec (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Automacs pro separator, by Miltenyi Biotec (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Macs neutrophil, by Miltenyi Biotec (2 mentions)
Golgiplug, by BD (2 mentions)
Pkh26, by Merck Group (2 mentions)
Red blood cell lysis buffer, by Merck Group (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Lymphocyte separation medium, by MP Biomedicals (2 mentions)
Neutrophil isolation kit, by Miltenyi Biotec (2 mentions)
Mono poly resolving medium, by Sumitomo Pharma (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi complete rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Facscanto, by BD (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
79 protocols using monocyte isolation kit
1
Isolation of Immune Cells from Blood and Skin
2
Cellular Migration Assay for Monocyte Trafficking
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For the cellular migration assays tissue culture-treated 6.5 mm transwells with 5.0 μ m pore polycarbonate membrane inserts were placed in 24 well plates containing 1.25x106 TSEC or 5x106 AML12 cells either untreated or incubated with LPS (100ng/ml) and FFA (0.33uM PA acid and 0.33uM OA) and equilibrated for 1 hour. Bone marrow monocytes were isolated by negative bead selection from C57BL6 bone marrow using a monocyte isolation kit (Miltenyi). 0.5–1 x106 monocytes were added to the transwell in 100ul volume and incubated at 37C for 3 hours. Following incubation transwells were removed and the wells were harvested and stained with fluorescently labeled antibodies against CD45, CD11b, F480, Ly6G and Ly6C. 5x104 polystyrene/divinylbenzene beads (Bang Laboratories PS07N/9400) were added to the tube and 10,000 beads per sample were collected by flow cytometry. The total number of migrated cells was calculated by dividing the number of counted cells by the number of counted beads and multiplying this number by 50,000 (the total number of beads in the sample) [(# of counted cells/# of counted beads) x 50,000]. The chemotactic index was determined by dividing the number of migrated beads in the experimental wells by the number of migrated beads in the control wells.
3
Monocyte Isolation and Influenza Stimulation
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Bone marrow cells were obtained by mechanical flushing of femurs and tibias. Monocytes were directly isolated by negative selection using the monocyte isolation kit according to manufacturer’s instructions (Miltenyi Biotec; 130-100-629) and had a purity of >95%. Monocytes were seeded at 2 × 105 cells per well in 200 µl in a 96-well plate and rested for 2 h before stimulation with X31 (MOI 1) in the presence or absence of IRS661 (1 µM).
4
Monocyte Adoptive Transfer in Tumor Models
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Monocytes were purified from the bone marrow of C57Bl/6 Ly.1 (CD45.1) mice using the Monocyte Isolation Kit (Miltenyi). The enriched population contained >95% of Ly6Chigh monocytes. 1.5-2.0 × 106 purified Ly6Chigh monocytes were infused i.v. into Ccr2-/- CD45.2 mice bearing 3-day established LLC-Mock, LLC-SULT2B1b or LLC-mCherry tumors. Twenty-four and 72 hours later, mice were sacrificed, and tumors evaluated for weight and for the content of monocyte-DCs. For cell proliferation experiments, ccr2-/- mice (CD45.2+) were injected with 5 × 106 tumor cells. After 3 days, mice received CD45.1+ monocytes. Twenty-four hours later, mice were injected i.p. with 1 mg/mouse of EdU. Four hours later, mice were sacrificed, tumor collected and digested as reported above. Cells were then stained, run by FACScanto (BD) and analyzed with FlowJo software. For the analysis of draining lymph nodes, ccr2-/- mice (CD45.2+) were injected with 5 × 106 tumor cells. After 3 days, mice received CD45.1+ monocytes and 48 hours later lymph nodes were collected and treated as reported in immunofluorescence staining and confocal analysis section.
5
Evaluating Immunomodulatory Properties of CNCs
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Cytocompatibility and immunomodulatory properties of CNCs were tested in vitro using human peripheral blood mononuclear cells (PBMCs) and MoDCs. Before human blood sample collection, written informed consents were provided by health donors, in accordance with the Declaration of Helsinki and approvals by the Ethical Committee of the Institute for the Application of Nuclear Energy (INEP). PBMCs were isolated from buffy coats by density gradient centrifugation in LymphoprepTM gradient media (Axis Shield PoC AS, Oslo, Norway), according to the manufacturer’s instructions. Monocytes and T cells were purified from PBMCs by magnetic-activated cell sorting (MACS) using Monocyte Isolation Kit and Pan T cell Isolation Kit (all Miltenyi Biotec, Bergisch Gladbach, Germany), where LS column flow-through represented the enriched populations of CD14+ monocytes and CD3+ T cells, respectively.
6
Adoptive Transfer of Monocytes
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BM Ly6C+ monocytes were isolated from congenic CD45.1 wild-type mice using the Monocyte Isolation Kit (Miltenyi Biotec, 130-100-629). Around 2 × 106 BM Ly6C+ monocytes were administered i.v. into CD45.1/CD45.2 IMDTR mice that were injected i.p. with 50 ng DT 24 h before monocyte transfer to deplete endogenous IMs.
7
Bone Marrow Monocyte Enrichment
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BM CD11b+Ly6C+ monocyte enrichment was performed using the monocyte isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. Briefly, BM cells were suspended in MACS buffer and Fc receptor (FcR) blocking solution was added and incubated with a cocktail of biotinylated primary monoclonal antibodies at 2–8 °C for 5 min to label non-targets cells. Cells were washed once and secondary anti-biotin monoclonal antibodies conjugated to Microbeads were then added to cell suspension. After 10 min of incubation at 2–8 °C, cell suspensions were run in autoMACS ProSeparator (Miltenyi Biotec) and depletion program ‘’Depletes” was used throughout all experiments to deplete non-target cells.
8
Isolated Mouse Monocyte Attachment Assay
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Primary mouse monocytes were isolated from bone marrow of wild-type C57BL/6 male mice using the Monocyte Isolation Kit for mice according to manufacturer’s instructions (Miltenyi Biotec). Primary mouse monocytes were incubated with CellTracker Green CMFDA Dye for 30 min according to manufacturer’s instructions (Life Technologies) and used for monocyte attachment assay.
9
PBMC Isolation and P. acnes Stimulation
10
Generating M1 and M2 Macrophages from Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were obtained from blood-donor buffy coats (Etablissement Français du Sang Pyrénéees-Méditerranée [EFS]) after density gradient centrifugation. Human monocytes were isolated from PBMCs using “monocyte isolation kit” (Miltenyi). Monocytes were plated in RPMI supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum (Hyclone, Perbio) + 100 ng/mL macrophage colony-stimulating factor (Peprotech) for 7 days, then monocyte-differentiated MΦ were polarized for 24 hr into pro-inflammatory M1MФ using IFN-γ (20 ng/mL; Peprotech) + LPS (1 μg/mL; InvivoGen) or anti-inflammatory M2MФ using IL-4 (20 ng/mL; Peprotech).
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