Easysep magnetic nanoparticles

Manufactured by STEMCELL

Sourced in Canada

EasySep® Magnetic Nanoparticles are superparamagnetic particles designed for the rapid and efficient isolation of target cells from complex samples. They enable the magnetic separation of cells without the need for columns or specialized equipment.

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Lab products found in correlation

Easysep cd45 depletion cocktail, by STEMCELL (2 mentions) Easysep human cd25 positive selection cocktail kit, by STEMCELL (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Lymphocyte separation solution, by TBD Science (1 mentions) Easysep pe selection cocktail, by STEMCELL (1 mentions) Cytoflex, by Beckman Coulter (1 mentions) Easyeights easysep magnet, by STEMCELL (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Easysep magnet, by STEMCELL (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Fcr blocking reagent, by Miltenyi Biotec (1 mentions)

8 protocols using easysep magnetic nanoparticles

Purification of Tim-3+ CD4+ T Cells

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PBMCs were isolated from six patients with aGVHD using a lymphocyte separation solution (TBD Science). Subsequently, the Negative Selection CD4⁺ T‐cell sorting kit (STEMCELL Technologies) was used to enrich CD4⁺ T cells. The sorted CD4⁺T cells were re‐suspended to 5 × 10⁷/mL and incubated with Tim‐3‐PE antibody (BD, Clone:7D3, 2 µg/mL) in the dark for 15 min. Then, the incubation with 100 μL of EasySep® PE Selection Cocktail (STEMCELL Technologies) and 50 μL of EasySep® Magnetic Nanoparticles (STEMCELL Technologies) was performed. Finally, Tim‐3⁺CD4⁺ T cells were evaluated by flow cytometry, and their purity was over 90%.

Pang N., Tudahong S., Zhu Y., He J., Han C., Chen G., Wang W., Wang J, & Ding J. (2024). Galectin‐9 alleviates acute graft‐versus‐host disease after haplo‐hematopoietic stem cell transplantation by regulating regulatory T cell/effector T cell imbalance. Immunity, Inflammation and Disease, 12(2), e1177.

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Isolation and Quantification of CTCs

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Combined negative and positive selection, which had been designed and validated [35 (link)], was utilized for CTC isolation. Four milliliters (mL) of blood were used for CTC counting, with a further 4 mL used for quality control. The negative selection, i.e., enrichment, included depletion of red blood cells (RBCs) and white blood cells (WBCs). RBC depletion was done by lysis of RBCs within 24 h after sampling. WBC depletion was done by adding EasySep CD45 Depletion Cocktail (STEMCELL Technologies Inc., Vancouver, BC, Canada) at 25 μL/mL and EasySep Magnetic Nanoparticles (STEMCELL Technologies Inc., Vancouver, BC, Canada) at 50 μL/mL and Hoechst 33,342 (1:500 in washing solution; Thermo Scientific, Waltham, MA, USA) for the nuclear staining. The positive selection, i.e., purification, was done by EpCAM isotyping. CTCs were defined as cells that were negative for CD45 and positive both for epithelial cell adhesion molecule (EpCAM) and Hoechst. Flow cytometry using CytoFLEX flow cytometer (Beckman Coulter, San Diego, CA, USA) quantitatively identified CTCs and calculated their numbers.

Wu C.Y., Fu J.Y., Wu C.F., Hsieh M.J., Liu Y.H., Liu H.P., Hsieh J.C, & Peng Y.T. (2021). Malignancy Prediction Capacity and Possible Prediction Model of Circulating Tumor Cells for Suspicious Pulmonary Lesions. Journal of Personalized Medicine, 11(6), 444.

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Isolation of Human CD4+CD25+ T-Regulatory Cells

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From venous whole blood collected from all patients, T-regulatory (T-reg) lymphocytes were isolated. The principle of isolation of CD4/CD25 cells from blood is based on the protocol of two-step selection of T-regulatory lymphocytes (T-reg), according to Sugiyama H. et al., 2005 [41 (link)] (with minor modifications and the use of kits StemCell Technologies, Vancouver, BC, Kanada). Firstly, CD4+ cells were obtained by negative selection using the RosetteSep^®Humane CD4+ T Cell Enrichment Cocktail kit (StemCell Technologies), according to the manufacturer’s instructions. Following this, CD25+ T lymphocytes were positively selected from enriched CD4+ cells using the EasySep^® Human CD25 Positive Selection Cocktail kit (StemCell Technologies) and magnetic beads EasySep^® Magnetic Nanoparticles (StemCell Technologies). The positively-selected cells were resuspended in 1 × PBS and used for RNA isolation.

Kutwin M., Migdalska-Sęk M., Brzeziańska-Lasota E., Zelga P, & Woźniacka A. (2021). An Analysis of IL-10, IL-17A, IL-17RA, IL-23A and IL-23R Expression and Their Correlation with Clinical Course in Patients with Psoriasis. Journal of Clinical Medicine, 10(24), 5834.

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Isolation of CD45+ Cells from Tissue

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10⁸ cells from tissue digest were resuspended in 1mL of PEF buffer (1xPBS+ 2% FBS+ 1mM EDTA). Cells were incubated with Fc block (Bioledgend) for 5mins at room temperature. CD45 APC antibody (1.5μg/mL, Stem Cell Technologies) was added and cells were incubated at room temperature for 15mins. After this incubation period, the EasySep® APC selection cocktail (110μg/mL, Stem Cell Technologies) was added and cells were incubated for 15mins at room temperature. EasySep® magnetic nanoparticles (50μL/mL, Stem Cell Technologies) were mixed and cells were incubated for 10mins at room temperature. To positively select for CD45 expressing cells, PEF buffer was added to bring samples to 2.5mL and suspension was transferred to 5mL polystyrene tubes. Tubes were placed in an EasyEights™ EasySep™ Magnet (Stem Cell Technologies) and incubated at room temperature for 10 mins. With the tube still in the magnet, supernatant was removed as the first flow through to yield the CD45⁻ population. The tube was removed from the magnet and wash steps were repeated three times. After the final wash, the tube was removed from the magnet and the cells were resuspended in 2mL PEF buffer, yielding the CD45⁺ population.

Wilkinson M.L., Abramova E., Guo C., Gow J.G., Murray A., Koudelka A., Cechova V., Freeman B.A, & Gow A.J. (2020). Fatty acid nitroalkenes inhibit the inflammatory response to bleomycin-mediated lung injury. Toxicology and applied pharmacology, 407, 115236.

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Two-Stage T-Reg Cell Isolation from Blood

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In the venous whole blood collected from all patients, 2-stage selection of T-regulatory lymphocytes was performed. The first step was obtaining CD4+ cells using the RosetteSep ® Humane CD4+ T Cell Enrichment Coctail kit (StemCell Technologies, Vancouver, Canada), according to the manufacturer's protocol. In the second stage, CD25+ T lymphocytes were selected from enriched CD4+ cells using the EasySep ® Human CD25 Positive Selection Cocktail kit (StemCell Technologies, Vancouver, Canada) and magnetic beads EasySep ® Magnetic Nanoparticles (StemCell Technologies, Vancouver, Canada). The positively selected cells were resuspended in 1 × PBS and destined for RNA isolation.

Kutwin M., Migdalska-Sęk M., Brzeziańska-Lasota E., Zelga P, & Woźniacka A. (2020). Analysis of molecular markers as IL-12, IL-22 and IFN-γ in correlation with a clinical course in patients with psoriasis. International journal of occupational medicine and environmental health, 33(5).

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Isolation and CFSE-labeling of OT-II CD4+ T cells

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Single cell suspensions from the spleen and pooled lymph nodes (cervical, brachial, axillary, mesenteric and iliac lymph nodes) of OT-II transgenic mice were enriched for CD4 + T cells, by negative selection using the Easy-Sep magnetic nanoparticles (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer’s protocol. The purity of the CD4+ T cell population in the enriched fraction was > 95%, as determined by flow cytometric analysis. CD4 + isolated T cells were pooled and stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE, 7.5 μM, Invitrogen) [32 (link)], for 10 min at 37 ℃. An amount of 2.5 10⁶ of CFSE-labelled CD4+ T cells was injected into the tail vein of each recipient mouse.

Boianelli A., Pettini E., Prota G., Medaglini D, & Vicino A. (2015). A Stochastic Model for CD4+ T Cell Proliferation and Dissemination Network in Primary Immune Response. PLoS ONE, 10(8), e0135787.

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Isolation and Transfer of OTII CD4+ T Cells

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OT-II transgenic mice (B6.Cg-Tg(TcraTcrb)425Cbn/J) were euthanized, and spleens were removed by dissection. Tissues were mashed onto a cell strainer, and the cells obtained were pooled, washed twice in PBS solution, and resuspended in PBS at 1 × 10⁸ cells/ml. OTII CD4⁺ T cells were isolated by negative selection, using EasySep magnetic nanoparticles (StemCell Technologies), according to the manufacturer’s protocol. The purity of the CD4⁺ cell population in the enriched fraction was >95%, as determined by flow cytometry analysis. CD4⁺ isolated T cells were pooled and stained with CellTrace™ Violet (CTV; 5 μM Invitrogen) for 20 min at 37 °C. 4 × 10⁶ of CTV-labelled T cells were transferred by intraperitoneal (i.p.) injections to mice phase shifted to ZT7 or ZT19 (described in more detail in next section).

Cervantes-Silva M.P., Carroll R.G., Wilk M.M., Moreira D., Payet C.A., O’Siorain J.R., Cox S.L., Fagan L.E., Klavina P.A., He Y., Drewinski T., McGinley A., Buel S.M., Timmons G.A., Early J.O., Preston R.J., Hurley J.M., Finlay D.K., Schoen I., Javier Sánchez-García F., Mills K.H, & Curtis A.M. (2022). The circadian clock influences T cell responses to vaccination by regulating dendritic cell antigen processing. Nature Communications, 13, 7217.

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Magnetic Separation of CD45+ Cells

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Following RBC lysis, cell suspension was transferred to a round-bottom tube (Becton Dickinson). To every 10⁸ cells, 200 μL of FcR Blocking Reagent (Miltenyi Biotec) and 50 μL of EasySep^® CD45 Depletion Cocktail (StemCell) were added and incubated at room temperature for 30 minutes. For immunomagnetic labelling, EasySep^® Magnetic Nanoparticles (StemCell) was added at 100 μL/mL and mixed by pipetting. The suspension was incubated at room temperature for 15 minutes and the volume was adjusted to 2.5 mL. The tube was placed into an EasySep^® magnet (StemCell) for 10 minutes. Labeled cells (CD45+) were separated by decanting the supernatant (CD45−) into a new tube. RNA was extracted from the CD45+ and CD45− fractions using the RNeasy Mini Kit (Qiagen).

Liu X., Ledet E., Li D., Dotiwala A., Steinberger A., Feibus A., Li J., Qi Y., Silberstein J., Lee B., Dong Y., Sartor O, & Zhang H. (2016). A Whole Blood Assay for AR-V7 and ARv567es in Prostate Cancer Patients. The Journal of urology, 196(6), 1758-1763.

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