Easysep monocyte enrichment kit

Manufactured by STEMCELL

Sourced in France, Canada

The EasySep™ Monocyte Enrichment Kit is a laboratory tool used for the isolation and enrichment of monocytes from human peripheral blood mononuclear cells (PBMCs). The kit utilizes antibody-based magnetic separation technology to select and recover the desired monocyte cell population.

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10 protocols using easysep monocyte enrichment kit

Isolation and Purification of PBMCs, CD4+ T Cells, and Monocytes

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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE healthcare, Buckingham, UK) density gradient centrifugation. CD4⁺ CD25^- T cells were purified using EasySep^® CD4⁺ CD25^- enrichment kit (StemCell Technologies, Meylan, France) according to manufacturer’s instructions. Monocytes were purified using EasySep^® monocyte enrichment kit (StemCell Technologies, Meylan, France) according to the manufacturer’s instructions.

Soskic B., Jeffery L.E., Kennedy A., Gardner D.H., Hou T.Z., Halliday N., Williams C., Janman D., Rowshanravan B., Hirschfield G.M, & Sansom D.M. (2021). CD80 on Human T Cells Is Associated With FoxP3 Expression and Supports Treg Homeostasis. Frontiers in Immunology, 11, 577655.

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Isolation of Human Monocytes and DCs

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The isolation of human monocytes and dendritic cells from de-identified human subjects was performed under IRB Protocol #2015-2437 and through the UC Irvine ICTS Blood Donor Program. Donor sex: CD14 M = 2 males, 3 females. CD16 M = 2 males, 2 females. Blood DCs were all from female donors. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-paque (GE Healthcare) gradient separation. In brief, blood was layered on top of Ficoll-paque and centrifuged in swinging bucket rotator without brake (400 × g, 40 minutes, 18°C). After centrifugation, plasma and upper layers were removed and PBMCs isolated from the interphase. Cells were then washed once with ice-cold PBS and used immediately. CD14 and CD16 monocytes were isolated via negative selection from PBMCs using the EasySep™ Monocyte Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Blood dendritic cells were isolated via negative selection from PBMCs using the EasySep™ Human Pan-DC Pre-Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Isolated cells were washed three times with PBS and sorted by FACS for either RNA-sequence analysis or used for further macrophage differentiation.

Abud E.M., Ramirez R.N., Martinez E.S., Healy L.M., Nguyen C.H., Newman S.A., Yeromin A.V., Scarfone V.M., Marsh S.E., Fimbres C., Caraway C.A., Fote G.M., Madany A.M., Agrawal A., Kayed R., Gylys K.H., Cahalan M.D., Cummings B.J., Antel J.P., Mortazavi A., Carson M.J., Poon W.W, & Blurton-Jones M. (2017). iPSC-derived human microglia-like cells to study neurological diseases. Neuron, 94(2), 278-293.e9.

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Culturing Diverse Cell Lines and Primary Macrophages

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MDA-MB-231 human breast carcinoma cells expressing GFP (MDA231) were kindly provided by Dr. Frank Gertler, MIT. PC3 human prostate carcinoma cells (PC3), MDA-MB-435S human melanoma cells (MDA435), and Raw 264.7 mouse macrophages (Raw) were obtained from American Type Culture Collection. MDA231, MDA435, and Raw cells were cultured in DMEM. PC3 were cultured in RPMI. All media were supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin/streptomycin. Cell lines were authenticated using Short Tandem Repeat profiling (Promega).
To generate primary bone marrow-derived macrophages (BMDM), bone marrow cells were first isolated from the femurs of C57BL/6 mice. These cells were then differentiated in RPMI supplemented with 10% FBS, 1% HEPES, 40 ng/mL MCSF (Peprotech) and 50 µM β-Mercaptoethanol for 7 days to produce BMDM. Primary human monocyte-derived macrophages (MDMΦ) were generated from monocytes isolated from whole blood (Research Blood Component) using a Ficoll-Paque gradient and the EasySep™ Monocyte Enrichment Kit (StemCell Tech.). These cells were cultured with IMDM supplemented with 2% L-glutamine and AB serum for 7 days to generate MDMΦ. All cells were cultured in a humidified incubator at 5% CO₂ and 37 °C.

Li R., Hebert J.D., Lee T.A., Xing H., Boussommier-Calleja A., Hynes R.O., Lauffenburger D.A, & Kamm R.D. (2016). Macrophage-secreted TNFα and TGFβ1 Influence Migration Speed and Persistence of Cancer Cells in 3D Tissue Culture via Independent Pathways. Cancer research, 77(2), 279-290.

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Macrophage Differentiation and Cytokine Profiling in GCA

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Monocytes were isolated from thawed PBMCs from GCA patients and healthy controls using an EasySep monocyte enrichment kit without CD16 depletion (StemCell Technologies). Isolated monocytes were cultured in the presence of GM‐CSF or macrophage colony‐stimulating factor (M‐CSF) to generate GM‐CSF–skewed or M‐CSF–skewed macrophages. Supernatants were collected for Luminex assay or enzyme‐linked immunosorbent assay (ELISA) (see the Supplementary Methods for details on the Luminex assay and ELISA methods). For the assessment of YKL‐40 levels, macrophages were activated by the addition of lipopolysaccharide (LPS) on day 5, and the supernatants were collected on day 7. Assessment of IL‐6 was included as stimulation control. For YKL‐40 and MMP‐9 kinetics experiments, GM‐CSF–skewed macrophages were generated and the supernatants were collected every 48 hours, with the final collection on day 8 for ELISAs.

van Sleen Y., Jiemy W.F., Pringle S., van der Geest K.S., Abdulahad W.H., Sandovici M., Brouwer E., Heeringa P, & Boots A.M. (2021). A Distinct Macrophage Subset Mediating Tissue Destruction and Neovascularization in Giant Cell Arteritis: Implication of the YKL‐40/Interleukin‐13 Receptor α2 Axis. Arthritis & Rheumatology (Hoboken, N.j.), 73(12), 2327-2337.

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Monocyte-derived Macrophage Activation Protocol

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Giant cell arteritis and HC monocytes were isolated from thawed PBMCs by negative selection using the EasySep monocyte enrichment kit (Stemcell, Vancouver, BC, Canada), which does not deplete CD16⁺ monocytes. Isolated monocytes were analysed by flow cytometry or cultured for 7 days in DMEM containing 2 mm glutamine, 60 µg mL⁻¹ penicillin–streptomycin and 10% FCS in the presence of 100 ng mL⁻¹ G M‐CSF (Peprotech, Rocky Hill, CT, USA) to generate GM‐MØs or 100 ng mL⁻¹ M‐CSF (Peprotech) to generate M‐MØs. The medium was replaced on the second and fourth day. On day 7, after collecting the supernatants for Luminex assay, monocyte‐derived macrophages were harvested using citrate saline (135 mm potassium chloride, 15 mm sodium citrate and 1 mm EDTA) for 15 min at 37°C. For macrophage activation experiments, monocytes were cultured for 7 days in the same medium in the presence of G M‐CSF/M‐CSF. The medium was replaced on the third and fifth days. LPS (100 ng mL⁻¹) (Sigma‐Aldrich, St. Louis, MO, USA) was added to the culture during the fifth day medium change. On day 7, supernatants were collected for ELISA.

Jiemy W.F., van Sleen Y., van der Geest K.S., ten Berge H.A., Abdulahad W.H., Sandovici M., Boots A.M., Heeringa P, & Brouwer E. (2020). Distinct macrophage phenotypes skewed by local granulocyte macrophage colony‐stimulating factor (GM‐CSF) and macrophage colony‐stimulating factor (M‐CSF) are associated with tissue destruction and intimal hyperplasia in giant cell arteritis. Clinical & Translational Immunology, 9(9), e1164.

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Isolation and Stimulation of Human Monocytes

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Primary human monocytes were freshly isolated from peripheral blood mononuclear cells (PBMCs) from individual donors using the EasySep Monocyte Enrichment kit from Stem Cell Technologies (negative selection). Cells underwent analysis by flow cytometry for purity (CD14-PE) (Becton Dickerson), with donor preps having cell purity greater than 90% for deep sequencing and further validation. Monocytes were suspended at 10 million/ml in X-Vivo medium (Lonza) supplemented with 5% AB human serum (Invitrogen) and incubated for 6 or 24 hours with either medium only or with media containing 100 ng/ml lipopolysaccharide (InVivoGen). All cells (both adherent and suspension) were harvested, washed, and pelleted, with the cell pellet snap-frozen on dry ice and placed at -80°C until further analysis.

Lu D., Yamawaki T., Zhou H., Chou W.Y., Chhoa M., Lamas E., Escobar S.S., Arnett H.A., Ge H., Juan T., Wang S, & Li C.M. (2019). Limited differential expression of miRNAs and other small RNAs in LPS-stimulated human monocytes. PLoS ONE, 14(3), e0214296.

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Monocyte Enrichment and Activation

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The EasySep Monocyte Enrichment Kit (StemCell) was used for this process following manufacturer’s recommended protocols. Briefly, bone marrow was harvested from both femurs of a C57/B6 mouse using a total of 4 ml of “recommended medium” (Phosphate buffered saline) + 2% FBS. Cells were pelleted and a negative selection biotinylated monocyte enrichment antibody cocktail was used. Samples were washed and incubated with a tetrameric antibody complex and magnetic dextran iron particles. An EasySep magnet was then used to separate the monocytes. Manufacturer’s state greater than 85% resulting population is comprised of monocytes after concentration.
Monocytes were exposed to 50 ng/ml of TNFα for 30 min to activate them and labelled with Calcein-AM, washed and immediately used for static incubation studies.

Kadam A.A., Gersch R.P., Rosengart T.K, & Frame M.D. (2019). Inflammatory monocyte response due to altered wall shear stress in an isolated femoral artery model. Journal of Biological Methods, 6(1), e109.

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Kidney and Bone Marrow Cell Isolation

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Kidney were isolated, released from the renal capsula, minced by scalpel and digested with Liberase TM (Roche) for flow cytometry analysis or with Collagenase Type 2 (Worthington) for kidney stromal cell cultures. Homogenized kidneys were incubated at 37 °C with stirring for 2 × 10 min with Liberase (0.65 WU/ml) in RPMI1640 (Invitrogen) or for 2 × 20 min with Collagenase Type 2 (400 UI/ml) in RPMI1640 10% heat inactivated fetal calf serum (FCS, PAA). Cell suspensions were obtained by successive filtration on 100 µm and 40 µm cell strainer filters (BD Falcon) after hypotonic lysis of red blood cells by ammonium chloride (0.83%).
Bone-marrow cells were collected by flushing the femurs and tibia of CX3CR1^gfp/+ CD45.1 C57BL/6 mice with 2 ml FACS buffer (DPBS, 4% FCS, 2 mM EDTA). Cell suspensions were filtrated on 100 µm cell strainer filters after lysis of red blood cells, centrifuged and counted for further analysis. Monocytes were enriched by negative selection from bone-marrow cell suspensions using EasySep^™ Monocyte enrichment kit (Stemcell Technologies).

Lefèvre L., Iacovoni J.S., Martini H., Bellière J., Maggiorani D., Dutaur M., Marsal D.J., Decaunes P., Pizzinat N., Mialet-Perez J., Cussac D., Parini A, & Douin-Echinard V. (2020). Kidney inflammaging is promoted by CCR2+ macrophages and tissue-derived micro-environmental factors. Cellular and Molecular Life Sciences, 78(7), 3485-3501.

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Alveolar Macrophage Cytokine Induction by NETs

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Human alveolar macrophages were isolated from transplant BAL of donor
lungs approximately 2 hours after reperfusion by plastic adherence in DMEM
(Gibco) complete medium supplemented with 20% human AB serum (Corning) and 20
ng/ml human M-CSF-1 (Peprotech) for 48 hours before the addition of NET
fragments. Following stimulation with NET preparations for 24 hours supernatant
cytokine accumulation was measured for indicated cytokines with ProCarta Luminex
assays (ThermoFisher) in accordance with manufacturer’s recommendations.
To generate human monocyte-derived dendritic cells, peripheral blood monocytes
were isolated with the Easy Sep monocyte enrichment kit (Stemcell) from healthy
human volunteers. 5 × 10⁶ monocytes per ml were added in
6-well plates and cultured in DMEM complete medium supplemented with 10% AB
serum, human GM-CSF (100ng/ml; Peprotech) and IL-4 (50 ng/ml; Peprotech) for 6
days with refreshment of media every 2 days prior to use in experiments.

Scozzi D., Wang X., Liao F., Liu Z., Zhu J., Pugh K., Ibrahim M., Hsiao H.M., Miller M.J., Yizhan G., Mohanakumar T., Krupnick A.S., Kreisel D, & Gelman A.E. (2018). Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 19(4), 1011-1023.

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Phagocyte Depletion and Adoptive Transfer

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Native phagocytes were depleted with clodronate liposomes (ClodronateLiposomes.org), which were administered for three consecutive days, and mice received 200 μl of clodronate liposomes (about 50 mg per mouse) according to the manufacturer’s instructions. BMDMs (1 × 10⁶) were adoptively transferred 24 hours after completion of clodronate treatment, and LPS was given the next day. Mice were treated with zymosan as described above. Monocytes were purified using the EasySep Monocyte Enrichment Kit from Stem Cell Technologies (19761) according to the manufacturer’s instructions. Monocytes (1 × 10⁶) were transferred into recipient mice, and zymosan was administered simultaneously.

Bennett L.F., Liao C., Quickel M.D., Yeoh B.S., Vijay-Kumar M., Hankey-Giblin P., Prabhu K.S, & Paulson R.F. (2019). Inflammation induces stress erythropoiesis through heme-dependent activation of SPI-C. Science signaling, 12(598), eaap7336.

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