Cd16 monocyte isolation kit

Manufactured by Miltenyi Biotec

The CD16+ Monocyte Isolation Kit is a laboratory product designed to isolate CD16+ monocytes from human peripheral blood mononuclear cells. It utilizes magnetic separation technology to selectively enrich for the target cell population.

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Lab products found in correlation

Cd14 microbead, by Miltenyi Biotec (2 mentions) Leucosep, by Greiner (1 mentions) Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions) Magnetic bead purification, by Miltenyi Biotec (1 mentions) Ficoll, by GE Healthcare (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions) Anti cd14 microbeads, by Miltenyi Biotec (1 mentions)

8 protocols using cd16 monocyte isolation kit

Isolation of Monocyte Subsets

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The experiments conform to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report and were approved by the local ethics board. Human CD14⁺⁺CD16^neg and CD14⁺CD16⁺⁺ monocytes were isolated from blood rings of healthy donors after written consent (Blood bank, Medizinische Hochschule Hannover) using CD14 microbeads and CD16 monocyte isolation kit (Miltenyi Biotec), respectively, according to manufacturer's instructions. The purity of the isolated cells was more than 90% routinely tested by flow cytometry. Human aortic endothelial cells (HAEC) were purchased from Lonza and cultured as per manufacturer's instructions.

Getzin T., Krishnasamy K., Gamrekelashvili J., Kapanadze T., Limbourg A., Häger C., Napp L.C., Bauersachs J., Haller H, & Limbourg F.P. (2017). The chemokine receptor CX 3 CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury. EMBO Molecular Medicine, 10(2), 151-159.

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Isolation of CX3CR1-expressing CD16+ Monocytes

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Human primary monocytes (5 × 10⁶/mL) were cultured in 5 mL RPMI medium containing 2% FCS in teflon coated bags [56 (link)] in the presence or the absence of recombinant human CX3CL1 (50 ng/mL, Biolegend) at 37 °C and 10% CO₂. After 18 h of culturing, monocytes were collected and subjected to isolation of CD16⁺ monocytes, using the CD16⁺ Monocyte Isolation Kit (Miltenyi Biotec), according to the manufacturer’s manual. The primary monocytes were subjected to a two-step procedure. First, cells were labeled with a non-monocyte depletion cocktail, for labeling and removing CD15⁺ granulocytes and CD56⁺ natural killer cells. Thereafter, the flow through fraction was labeled with CD16⁺ magnetic microbeads for positive selection of CD16⁺ monocytes. CD16 positive selection was performed to enrich CD16⁺ monocytes, described to express the CX3CL1-receptor CX3CR1 [23 (link),24 (link)]. CX3CR1 expression in CD16⁺ monocytes was confirmed by qPCR (Figure S3).

Nonn O., Güttler J., Forstner D., Maninger S., Zadora J., Balogh A., Frolova A., Glasner A., Herse F, & Gauster M. (2019). Placental CX3CL1 is Deregulated by Angiotensin II and Contributes to a Pro-Inflammatory Trophoblast-Monocyte Interaction. International Journal of Molecular Sciences, 20(3), 641.

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Purification of Human Monocytes

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Human monocytes were isolated from the blood of healthy individuals as approved by the ethical committee of Hannover Medical School. Written consent was obtained before blood collection. Cells were purified using CD16⁺ monocyte isolation kit (Miltenyi Biotec) according to the manufacturer's instruction. CD14⁺ monocytes were retrieved in subsequent purification step from CD16^neg fraction using CD14 microbeads.

Gamrekelashvili J., Giagnorio R., Jussofie J., Soehnlein O., Duchene J., Briseño C.G., Ramasamy S.K., Krishnasamy K., Limbourg A., Häger C., Kapanadze T., Ishifune C., Hinkel R., Radtke F., Strobl L.J., Zimber-Strobl U., Napp L.C., Bauersachs J., Haller H., Yasutomo K., Kupatt C., Murphy K.M., Adams R.H., Weber C, & Limbourg F.P. (2016). Regulation of monocyte cell fate by blood vessels mediated by Notch signalling. Nature Communications, 7, 12597.

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Isolation and Cryopreservation of Peripheral Blood Mononuclear Cells

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Peripheral Blood Mononuclear Cells (PBMCs) were immediately isolated by density centrifugation using pre-filled Greiner Bio-One Leucosep® tubes according to the manufacturer’s recommendations. PBMCs rings were collected and washed twice in PBS before final suspension at 20 × 10⁶ cells/ml in ice-cold freezing medium (50% RPMI-1640, 40% fetal bovine serum, 10% DMSO) and transferred at −80°C in Nalgene® Mr. Frosty for short-term storage (<1 month). For monocyte isolation, 40 × 10⁶ to 60 × 10⁶ cells of cryopreserved PBMCs were thawed and quickly washed with 13 ml ice-cold RPMI-1640. Median cell viability upon recovery was assessed by trypan blue exclusion (88.37%, IQR: 82.1-97). Pan- and CD16+ monocyte purification were performed using Pan Monocyte Isolation Kit and CD16+ Monocyte Isolation Kit respectively according to manufacturer’s instructions (Miltenyi Biotec GmbH).

Portevin D., Pflüger V., Otieno P., Brunisholz R., Vogel G, & Daubenberger C. (2015). Quantitative whole-cell MALDI-TOF MS fingerprints distinguishes human monocyte sub-populations activated by distinct microbial ligands. BMC Biotechnology, 15, 24.

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Purification of Monocyte Subsets

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PBMCs were separated by Ficoll (GE Healthcare, Port Washington, NY) density gradient centrifugation and subjected to cell subset purification by magnetic bead purification (all from Miltenyi Biotec, Auburn, CA). CD4+ T cells and total monocytes were purified using CD4+ T cell isolation kit and CD14 microbeads, respectively (purity>95% for both) following manufacturer’s instructions. For purification of CD16+ and CD16⁻ monocyte subsets, the CD16+ monocyte isolation kit (Miltenyi Biotec) was used first to purify CD16+ monocytes by positive selection (purity>95%) and the negatively selected fraction was then incubated with CD14 microbeads to obtain the CD14⁺CD16⁻ cell population (purity>95%) following manufacturer’s instructions.

Zhong H., Bao W., Friedman D, & Yazdanbakhsh K. (2014). Hemin controls T cell polarization in sickle cell alloimmunization. Journal of immunology (Baltimore, Md. : 1950), 193(1), 102-110.

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CFSE Labeling of CD16+ Monocytes

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Peripheral blood mononuclear cells were separated over Ficoll-Paque as above and the CD16⁺ monocytes were then magnetically isolated using a CD16⁺ monocyte isolation kit (Miltenyi Biotec) according to manufacturer's instructions. CD16⁺ monocytes were then washed in phosphate buffered solution (PBS) and incubated in PBS containing 2.5 μM Carboxyfluorescein succinimidyl ester (CFSE [Biolegend]) for 20 min at 37°C. Cells were then washed in RPMI 1640 containing penicillin/streptomycin, L-glutamine and 10% HI-FCS and added back into non-labeled PBMCs from the same donor. PBMCs were then seeded in 12 well plates in complete RPMI and incubated with or without 20 ng/mL LPS for 3 h prior to identification by flow cytometry, as above.

Waller K., James C., de Jong A., Blackmore L., Ma Y., Stagg A., Kelsell D., O'Dwyer M., Hutchins R, & Alazawi W. (2019). ADAM17-Mediated Reduction in CD14++CD16+ Monocytes ex vivo and Reduction in Intermediate Monocytes With Immune Paresis in Acute Pancreatitis and Acute Alcoholic Hepatitis. Frontiers in Immunology, 10, 1902.

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Purification and Isolation of Platelets and Monocytes from RA Patients

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The study protocols were approved by the institutional review board (IRB) of Seoul National University Hospital and Chungnam National University Hospital. Peripheral blood of RA patients and healthy controls (HCs) was drawn after obtaining written, informed consent. The methods were performed in accordance with the approved guidelines. The patient characteristics of RA patients enrolled in this study are summarized in Table 1. To obtain platelets, platelet-rich plasma (PRP) was prepared from whole blood by centrifugation at 190 g for 15 min at room temperature (RT). Subsequently, platelet pellet was prepared from PRP by centrifugation at 2,400 g for 5 min and was resuspended with 25mM HEPES-buffered Tyrode’s solution (Sigma-Aldrich, St. Louis, MO). Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation (Bicoll separating solution; BIOCHROM Inc., Cambridge, UK). To purify CD14⁺CD16^- monocytes, CD16⁺ monocytes were negatively depleted from PBMC with CD16⁺ Monocyte Isolation Kit (Miltenyi Biotec Inc., Auburn, CA) and CD14⁺ monocytes were positively purified from CD16⁺ cell-depleted PBMC using anti-CD14 microbeads (Miltenyi Biotec Inc.).

Lee S.J., Yoon B.R., Kim H.Y., Yoo S.J., Kang S.W, & Lee W.W. (2021). Activated Platelets Convert CD14+CD16- Into CD14+CD16+ Monocytes With Enhanced FcγR-Mediated Phagocytosis and Skewed M2 Polarization. Frontiers in Immunology, 11, 611133.

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Isolation of Monocyte Subsets from Blood

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Peripheral blood mononuclear cells were isolated from buffy coats obtained from the National University Hospital Blood Transfusion Services, Singapore. Informed written consent was given in accordance to the declaration of Helsinki. All blood samples and procedures were approved by the NHG Domain Specific Review Board, Singapore (Reference code 08-352E). Following Ficoll-Hypaque density gradient centrifugation of the buffy coat, CD16 À and CD16 + monocyte subsets were isolated using the CD16 Monocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions, with some modifications. Briefly, after magnetic depletion of natural killer cells, granulocytes, B cells and T cells using anti-CD56, anti-CD15, anti-CD3 and anti-CD19 microbeads, the CD16 + monocytes were positively selected using anti-CD16 microbeads. The CD16 À monocytes were then isolated from the negative fraction with anti-CD14 microbeads. Purity of the monocyte subsets obtained was assessed by flow cytometry with fluorochrome-conjugated anti-CD14 and anti-CD16 antibodies and was consistently ≥ 95% (see Supporting information, Fig. S1). The percentage of dead cells in the isolated subsets was always < 5%.

Dang T.M., Wong W.C., Ong S.M., Li P., Lum J., Chen J., Poidinger M., Zolezzi F, & Wong S.C. (2015). MicroRNA expression profiling of human blood monocyte subsets highlights functional differences. Immunology, 145(3).

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