Easysep positive selection kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep positive selection kit is a lab equipment product designed for the isolation and enrichment of target cells from complex samples. It utilizes magnetic particles and an easy-to-use protocol to selectively separate desired cell populations.

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Lab products found in correlation

Mouse cd25 positive selection kit, by STEMCELL (1 mentions) Nih3t3, by American Type Culture Collection (1 mentions) E0771, by American Type Culture Collection (1 mentions) Monocyte enrichment kit, by STEMCELL (1 mentions) Ficoll paque gradient, by GE Healthcare (1 mentions) Easysep mouse cd8 t cell isolation kit, by STEMCELL (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Rneasy micro kit, by Qiagen (1 mentions) Iscan array scanner, by Illumina (1 mentions) Encore biotinil module, by Tecan (1 mentions) Ovation pico wta systems v2, by Tecan (1 mentions) Ingenuity pathway analysis ipa, by Qiagen (1 mentions) Genomestudio, by Illumina (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

7 protocols using easysep positive selection kit

Mouse T cell and Tumor Cell Isolation

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Mouse splenic CD4⁺ or CD8⁺ T cells were purified from C57BL/6 mice by EasySep positive selection kit (StemCell Technologies). For naturally occurring mouse regulatory T cell (nTreg) purification, CD4⁺ T cells were negatively purified by mouse CD4⁺ T cell pre-enrichment kit (StemCell Technologies) and followed to purify CD4⁺CD25⁺ cells by mouse CD25 positive selection kit (StemCell Technologies). The mouse tumor cell lines E0771, LL/2 and B16F10, and embryonic fibroblasts cell line NIH/3T3 were all obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). All mouse cells were cultured in medium containing complete RPMI 1640 medium, 10%(v/v) FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Liu X., Si F., Bagley D., Ma F., Zhang Y., Tao Y., Shaw E, & Peng G. (2022). Blockades of effector T cell senescence and exhaustion synergistically enhance antitumor immunity and immunotherapy. Journal for Immunotherapy of Cancer, 10(10), e005020.

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Isolation of Murine Macrophages and Monocytes

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Detailed protocols are provided as Supplementary Information. In brief, macrophages were isolated using magnetic beads with an EasySep positive selection kit (STEMCELL Technologies Inc., Vancouver, Canada). Monocytes were isolated from whole blood using a monocyte enrichment kit (STEMCELL). Bone-marrow derived monocytes were enriched in a similar fashion from cells isolated from perfused femurs and tibias of eight-week old transgenic B6.Cg-Tg (ACTB-mRFP1)1F1Hadj/J mice (termed RFP) male mice and matched wild-type controls.

Cortese R., Gileles-Hillel A., Khalyfa A., Almendros I., Akbarpour M., Khalyfa A.A., Qiao Z., Garcia T., Andrade J, & Gozal D. (2017). Aorta macrophage inflammatory and epigenetic changes in a murine model of obstructive sleep apnea: Potential role of CD36. Scientific Reports, 7, 43648.

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Isolation and Characterization of Human CD14+ Monocytes

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Blood from patients or healthy donors was diluted 1:1 in phosphate-buffered saline (PBS), layered onto Ficoll-Paque gradients with a density of 1.078 g/ml (GE Healthcare, United Kingdom), and centrifuged for 15 min at 800 × g. The mononuclear cells at the interface were aspirated, washed, and resuspended at 3 × 10⁵ cells/well in complete RPMI medium containing 10% fetal calf serum (FCS). CD14⁺ cells were purified using the EasySep positive selection kit according to the manufacturer’s protocol (StemCell Technologies, Canada). Cells were stained with anti-CD14 (allophycocyanin [APC]) (eBioscience, San Diego, CA) and anti-CD16 (phycoerythrin [PE]) (Becton, Dickinson, Franklin Lakes, NJ), and purity was checked by fluorescence-activated cell sorting (FACS).

Hirako I.C., Gallego-Marin C., Ataide M.A., Andrade W.A., Gravina H., Rocha B.C., de Oliveira R.B., Pereira D.B., Vinetz J., Diamond B., Ram S., Golenbock D.T, & Gazzinelli R.T. (2015). DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients. mBio, 6(6), e01605-15.

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CD8+ T Cell Proliferation Assay with Suppressive Myeloid Cells

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We used either aaMϕ or MDSCs sorted out of B16-OVA tumors. CD8⁺ T cells were isolated from OT1+/+Thy1.1+/+ mice using the EasySEP mouse CD8⁺ T cell isolation kit (StemCell Technologies cat. # 19853). CD11c dendritic cells (DCs) were isolated from the spleen using anti-CD11c biotin antibody and the EasySEP positive selection kit (Stem Cell technologies, cat. # 18559). DCs were pulsed with OVA _257-268 peptide (Sigma, S7951) for 6 hrs. T cells were labeled with CFSE at a final concentration of 5 μM. They were then incubated with OVA-pulsed DCs at a 1:10 ratio in the presence or absence of suppressive myeloid cells (MDSCs or aaMϕ). T cell proliferation was determined by detecting CFSE dilution on gated CD3⁺CD8⁺Thy1.1⁺ cells by flow cytometry.

Halaby M.J., Hezaveh K., Lamorte S., Ciudad M.T., Kloetgen A., MacLeod B.L., Guo M., Chakravarthy A., Medina T.D., Ugel S., Tsirigos A., Bronte V., Munn D.H., Pugh T.J., DeCarvalho D.D., Butler M.O., Ohashi P.S., Brooks D.G, & McGaha T.L. (2019). GCN2 drives macrophage and MDSC function and immune suppression in the tumor microenvironment.. Science immunology, 4(42), eaax8189.

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Isolation and Activation of Monocytes/Macrophages

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Spleens were removed and ground to pass them through a 50 μm nylon mesh (BD Falcon, Bedford, MA, USA). The prepared single-cell suspension was washed using RPMI1640 (Invitrogen Co., CA, USA) and was counted. Then, the cells were re-suspended (1 × 10⁶ cells/mL) for cell sorting. CD11b⁺ monocytes/macrophages were isolated using an EasySep Positive Selection Kit (STEMCELL Technologies Inc., Canada) according to the manufacturer’s instructions. The purified monocytes or macrophages were cultured in culture dishes with 50 ng/mL of lipopolysaccharide (LPS) for 6 h [23 (link)].

Wang H., Deng Q.W., Peng A.N., Xing F.L., Zuo L., Li S., Gu Z.T, & Yan F.L. (2018). β-arrestin2 functions as a key regulator in the sympathetic-triggered immunodepression after stroke. Journal of Neuroinflammation, 15, 102.

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Transcriptional Profiling of IL-9R+ T Helper Cells

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Human CD4⁺ T cells were isolated from PBMCs by EasySep positive selection kit (Stemcell Technologies) according to the manufacturer’s instructions. Positively selected CD4⁺ T cells were stained for subsequent T_H cell subset sorting (see Materials and Methods, Isolation and purification of human T cell subsets from peripheral blood). After T cell single-cell cloning, cells were screened for IL-9R expression by flow cytometry. Skin biopsies from positive patch test reactions were cultured in culture medium and CD4⁺ T cells were isolated and screened for IL-9R expression by flow cytometry. Five IL-9R⁺ T_H clones isolated from blood and three IL-9R⁺ T_H cells isolated from skin biopsies, respectively, were incubated in presence or absence of recombinant human IL-9 (5 ng/ml) for 12 h. Total RNA was isolated with the RNeasy Micro Kit (Qiagen) according to manufacturer’s instruction and RNA-seq was performed.

Bertschi N.L., Steck O., Luther F., Bazzini C., von Meyenn L., Schärli S., Vallone A., Felser A., Keller I., Friedli O., Freigang S., Begré N., Radonjic-Hoesli S., Lamos C., Gabutti M.P., Benzaquen M., Laimer M., Simon D., Nuoffer J.M, & Schlapbach C. (2023). PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis. Nature Communications, 14, 2471.

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Transcriptomic Profiling of CD19+ B Cells in Sepsis

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CD19+ B cells from sepsis and healthy controls were isolated to a purity of >99% using EasySep ® Positive Selection kit (Stem Cell Technologies). Purity was confirmed with flow cytometry. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA was converted to cDNA using the Ovation ® Pico WTA Systems V2 and labelled using the Encore ® BiotinIL Module (Nugen). Labelled cDNA was hybridized onto Infinium Illumina HT12v4 arrays and data collected on an iSCAN array scanner (Illumina). Following scanning, basic QC statistics were completed, displaying hybridisation controls, background signal and mean gene intensity of all genes and those of housekeeping genes. Array results were compiled using Genome Studio (Illumina) following quantile normalisation. Differences in messenger RNA abundance between the sepsis and healthy controls were assessed using Partek Genomic Suite (Partek Inc.). Similarities and differences in cell specific gene expression pattern in health and sepsis were assessed using heat maps and the top canonical pathways identified using Ingenuity Pathway Analysis (IPA) (Qiagen). Apoptosis genes and pathways were also studied.

Shankar-Hari M., Fear D., Lavender P., Mare T., Beale R., Swanson C., Singer M, & Spencer J. (2017). Activation-Associated Accelerated Apoptosis of Memory B Cells in Critically Ill Patients With Sepsis. Critical care medicine, 45(5).

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