Cd2 microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

CD2 microbeads are a laboratory tool designed for the isolation and enrichment of CD2-positive cells. They consist of superparamagnetic particles coated with antibodies specific to the CD2 cell surface antigen. When cells expressing CD2 are incubated with these microbeads, they become magnetically labeled and can be separated using a magnetic field.

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Lab products found in correlation

Pe conjugated anti hcd2 ab, by BD (2 mentions) Facscalibur, by BD (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Macs system, by Miltenyi Biotec (1 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Il 15, by Thermo Fisher Scientific (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) B cell isolation kit 2, by Miltenyi Biotec (1 mentions) Ebioscience intracellular fixation permeabilisation buffer set, by Thermo Fisher Scientific (1 mentions) Whole skin dissociation kit, by Miltenyi Biotec (1 mentions) Dnase 1, by Roche (1 mentions) Dead cell removal kit, by Miltenyi Biotec (1 mentions) Collagenase p, by Roche (1 mentions) Human cd28 microbead kit, by Miltenyi Biotec (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Tissuefaxs system, by TissueGnostics (1 mentions)

8 protocols using cd2 microbeads

Isolation and Culture of CD133+ Cells

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CD133⁺ cells were isolated by magnetic-activated cell sorting (MACS) with a CD133 MicroBead Tumor Tissue Kit (Miltenyi Biotec, 130-100-857) using methods described previously (18 (link)). The cells were cultured in serum-free media for sphere formation assays. When we explored whether the cytokines had a role in the differentiation, 10 ng/mL of interleukin (IL)-2, IL-3, IL-6, or transforming growth factor beta (TGF-β) was added to the culture for 24 hours. CD133⁺ cells from clinical tumor specimens were isolated by flow cytometry (FCM). CD2⁺ and CD2⁻ cells were isolated by MACS with CD2 MicroBeads (Miltenyi Biotec, 130-091-114).

Jia M., Jia X., Zhang D., Liu W., Yi S., Li Z., Cong B., Ma C., Li S, & Zhang J. (2021). CD2+ T-helper 17-like cells differentiated from a CD133+ subpopulation of non-small cell lung carcinoma cells promote the growth of lung carcinoma. Annals of Translational Medicine, 9(8), 687.

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Generation of CD8+CD28- T cells

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Cultures for the in vitro generation of CD8⁺CD28⁻ T cells (From individual A) were set up as described previously [13 (link)]. Briefly, CD8⁺ T cells were purified from PBMCs of individual A (A-CD8⁺ T cells) by positive selection using the MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. APCs were isolated from HLA-A, −B, −DR mismatched individual B (B-APCs) by depletion of CD2⁺ cells using CD2 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometric analysis showed that the purity of sorted cell suspensions was ≥97%. Enriched CD8⁺ T cells (2 × 10⁶ cells) were stimulated with B-APCs (1 × 10⁶ cells) for 9 days in 24-well plates in 2 ml complete medium (RPMI 1640 supplemented with 15% fetal bovine serum) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in the presence of IL-2 (PeproTech, Rocky Hill, NJ, USA) at 20 U/ml, IL-7 (PeproTech, Rocky Hill, NJ, USA) at 50 ng/ml, IL-15 (PeproTech, Rocky Hill, NJ, USA) at 50 ng/ml in an incubator at 37 °C and a humidified, 5.5% CO₂ atmosphere. After 9 days’ incubation, CD28⁻ cells were further enriched by a negative selection strategy, using anti-CD28-PE antibody and anti-PE microbeads combination to remove CD28⁺ cells (Miltenyi Biotec, Bergisch Gladbach, Germany). These CD8⁺CD28⁻ T cells were used for evaluating their suppressive activity.

Liu G., Yu Y., Feng F., Zhu P., Zhang H., Zhang D., Feng X., Zhang Z, & Liu Y. (2020). Human CD8+CD28− T suppressor cells expanded by common gamma chain (γc) cytokines retain steady allospecific suppressive capacity in vivo. BMC Immunology, 21, 23.

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Activation and Detection of MR1-restricted T cells

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B cells were isolated from PBMC using B Cell Isolation Kit II, human (Miltenyi Biotec) and T cells from an allogenic donor were isolated using CD2 MicroBeads, human (Miltenyi Biotec) and cell isolation was performed using the Miltenyi Biotec MidiMACS™ separator following the manufacturer’s instructions. B cells were cultured with 5-OP-RU at indicated concentrations for 4 hr. The B cells were then washed twice prior to addition of BFA and allogenic (MHC miss-matched, as MR1 is monomorphic) T cells at 1:1 effector:target ratio and cultured for 18 hr. Cells were collected and stained with viability dye, prior to fixation and permeabilisation using the eBioscience™ Intracellular Fixation & Permeabilisation Buffer Set (Invitrogen). Antibodies diluted in permeabilisation buffer were then incubated for 45 min at room temperature.

Howson L.J., Awad W., von Borstel A., Lim H.J., McWilliam H.E., Sandoval-Romero M.L., Majumdar S., Hamzeh A.R., Andrews T.D., McDermott D.H., Murphy P.M., Nours J.L., Mak J.Y., Liu L., Fairlie D.P., McCluskey J., Villadangos J.A., Cook M.C., Turner S.J., Davey M.S., Ojaimi S, & Rossjohn J. (2020). Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1. Science immunology, 5(49), eabc9492.

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MACS Sorting of Human CD2+ Cells

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MACS sorting of human CD2 (hCD2) (+) cells was performed using CD2 Microbeads (130-091-114, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). To monitor the results of MACS sorting, cells were stained with phycoerythrin (PE)-conjugated anti-hCD2 Ab (347597, BD Life Sciences, San Jose, CA, USA) for 30 min at 4°C. Flow cytometry was performed on a FACSCalibur (BD Life Sciences), and data were analyzed using the FlowJo software (BD Life Sciences). enChIP-real-time PCR enChIP-real-time PCR was performed as previously described (13) . The primers used in this study were reported previously (1) .

Yuno M., Fujita T., Fujii H., & Nagata S. (2021). MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis.

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Isolation and Stimulation of Skin T Cells

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For freshly isolated (FI) T-cell stimulation experiments, Collagenase P (Roche, Basel, Switzerland) digestion of fresh human skin was used essentially as previously described (31 (link)). In brief, dermatome-cut skin specimens were digested in complete Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (complete RPMI) containing 1 mg/ml Collagenase P (Roche) at 37°C on a rotator for 3 h. Next, 200 U/ml DNase I (Roche) were added and incubated for 15 min at 37°C. The cell suspension was diluted with 10 mM EDTA/PBS and filtered through 100- and 40-µm cell strainers. Dead cells were eliminated using a Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and T cells were enriched (purity range: 91–98%) with CD2 microbeads (Miltenyi Biotec) according to manufacturer’s instructions. Next, either IL-9 staining was performed or cells were further stimulated. For flow-cytometric analysis of surface proteins on FI skin T cells, the Whole Skin Dissociation Kit (Miltenyi Biotec) was used according to manufacturer’s instructions to digest human skin.

Kienzl P., Polacek R., Reithofer M., Reitermaier R., Hagenbach P., Tajpara P., Vierhapper M., Gschwandtner M., Mildner M., Jahn-Schmid B, & Elbe-Bürger A. (2019). The cytokine environment influence on human skin–derived T cells. The FASEB Journal, 33(5), 6514-6525.

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MACS Sorting of Human CD2+ Cells

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Yuno M., Fujita T., & Fujii H. (2021). MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis.

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Isolation and Purification of T Cell Subsets

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Peripheral blood mononuclear cells were isolated from fresh whole blood of healthy donors using lymphocyte separation medium by density gradient centrifugation. CD8⁺ and CD4⁺ T cells were positively selected using CD8 or CD4 isolation kits according to the manufacturer’s instructions (MACS, Miltenyi Biotech). APCs were isolated from PBMCs by depletion of CD2⁺ cells using CD2 Microbeads (Miltenyi Biotech). The purity of resulting population was >95%. When culture of the CD8⁺ T cells for indicated days, CD28⁺ cells were removed by positive selection using human CD28 MicroBead Kit (Miltenyi Biotech, purity > 99%), and the flow-through cells were collected as CD28⁻ cells (purity > 95%). Again, purity of all isolated cells was confirmed by flow cytometry. Isolated CD4⁺ T cells were labeled with 0.5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) for 7 min at 37°C.

Feng F., Liu Y., Liu G., Zhu P., Zhu M., Zhang H., Lu X., Liu J., Luo X, & Yu Y. (2018). Human CD8+CD28− T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner. Frontiers in Immunology, 9, 1442.

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HBsAg Uptake Quantification in CD2- Cells

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CD2⁻ cells (non-T non-NK cells) were negatively isolated using CD2 microbeads (Miltenyi Biotec), according to manufacturer’s protocol. Subsequently, CD2⁻ cells were incubated for 2 h with or without recombinant HBsAg (10 μg ml⁻¹; adr subtype) at 37 °C incubator and antigen uptake was stopped by two times cold wash in PBS. Cells were fixed and stained for HBsAg, using a two-step biotin–strepavidin staining protocol as previously described32 (link). Cells were cytospinned onto Superfrost Plus slides (Thermo Scientific) using CYTO-TEK Cytocentrifuge (Sakura Finetek), sealed with ProLong Gold Antifade Reagent with DAPI (4',6-diamidino-2-phenylindole; Invitrogen) and HBsAg staining was visualized using TissueFAXS system (TissueGnostics). The exposure time for fluorescein isothiocyanate (FITC) filter (for HBsAg staining) on the microscope was adjusted based on the negative controls (healthy CB cells) and positive controls (healthy CB cells incubated with recombinant HBsAg), to minimize autofluorescence/background staining without compromising signal. The total number of HBsAg⁺ cells and the total number of DAPI-stained nuclei were manually counted in ten random high power fields ( × 20 magnification).

Hong M., Sandalova E., Low D., Gehring A.J., Fieni S., Amadei B., Urbani S., Chong Y.S., Guccione E, & Bertoletti A. (2015). Trained immunity in newborn infants of HBV-infected mothers. Nature Communications, 6, 6588.

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