Cd4 cd8 microbeads

Manufactured by Miltenyi Biotec

CD4/CD8 MicroBeads are magnetic particles coated with antibodies specific for the CD4 or CD8 surface antigens on T cells. These beads can be used to isolate and enrich CD4+ or CD8+ T cells from a heterogeneous cell population.

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CD4 microbeads (226 citations) CD8 microbeads (181 citations) CD4 and CD8 microbeads (29 citations) Anti-CD4 and anti-CD8 microbeads (17 citations) CD4 or CD8 microbeads (9 citations) Anti-CD4 or anti-CD8 microbeads (6 citations) REAlease® CD4/CD8 (TIL) MicroBead Kit, (2 citations) CD4 and CD8 antibody conjugated microbeads (2 citations) Anti-CD4- or anti–CD8-conjugated microbeads (2 citations) Anti-CD4 or anti-CD8-coated magnetic microbeads (2 citations)

6 protocols using cd4 cd8 microbeads

Isolation and Characterization of Primary Human T Cells

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Blood samples were obtained from healthy donors at the University of Pittsburgh, Pittsburgh, PA (UPCI 09-069/IRB991206). PBMCs were isolated according to standard protocols and as previously described in detail (60 (link)). Briefly, blood samples from healthy volunteers (30 to 40 ml) were drawn into heparinized tubes and centrifuged on Ficoll-Hypaque gradients (GE Healthcare Bioscience, Chicago, IL). PBMCs were recovered, washed in AIM-V medium (Invitrogen), and immediately used for experiments. Naive CD4⁺ and CD8⁺ T cells were magnetically isolated using CD4⁺/CD8⁺ microbeads (Miltenyi, San Diego, CA) and used for subsequent experiments.

Yerneni S.S., Werner S., Azambuja J.H., Ludwig N., Eutsey R., Aggarwal S.D., Lucas P.C., Bailey N., Whiteside T.L., Campbell P.G, & Hiller N.L. (2021). Pneumococcal Extracellular Vesicles Modulate Host Immunity. mBio, 12(4), e01657-21.

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Enrichment and Activation of Antigen-Specific T Cells

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T cells were positively enriched using LS columns and CD4/CD8 MicroBeads according to the manufacturer’s protocol (Miltenyi Biotec). Isolated T cells were cultured in serum-free or serum-containing media in the presence or absence of cytokines. Subsequently, T cells were stimulated with either Expamers or Dynabeads Magnetic Beads (Thermo Fisher Scientific) according to in-house S.O.P. or manufacturer’s recommendations, respectively. Activated T cells were kept in culture for at least 7 days unless indicated otherwise.
For antigen-specific activation, Strep-tagged pMHCs were produced as described (Knabel et al., 2002). To generate pMHC/CD28 Expamers, Streptagged pMHC (HLA-A*02:01/pp65495-503) and anti-CD28 Fab fragment were added to the Strep-Tactin multimer backbone and complexed for 15 min at RT. Subsequently, pMHC/CD28 Expamers were added to PBMCs of an HLA-A*02:01⁺ CMV seropositive donor. Cells were checked before and after 8 days in vitro cell culture for the presence of HLA-A*02:01/pp65495-503 specific T cells by flow cytometry staining with fluorescently labeled pMHC multimer and anti-CD8 (PE; clone HIT8a) antibody.

Poltorak M.P., Graef P., Tschulik C., Wagner M., Cletiu V., Dreher S., Borjan B., Fraessle S.P., Effenberger M., Turk M., Busch D.H., Plitzko J., Kugler D.G., Ragan S., Schmidt T., Stemberger C, & Germeroth L. (2020). Expamers: a new technology to control T cell activation. Scientific Reports, 10, 17832.

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Isolation and Differentiation of Immune Cells

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PBMCs were isolated by Ficoll-Paque Plus (Cytiva Europe GmbH) density gradient centrifugation and then washed twice in culture medium (RPMI-1640 supplemented with 10% FBS) [30 (link)]. T cell isolation was performed through immunomagnetic cell sorting using CD4/CD8 microbeads (MACS Miltenyi Biotec). Human monocytes isolation was performed through immunomagnetic cell sorting using CD14 microbeads (MACS Miltenyi, Biotec) according to the manufacturer instructions.
For macrophage differentiation CD14⁺ monocytes (1 × 10⁶/ml) from healthy PBMCs were seeded in RPMI-1640 medium, 100 ng/ml M-CSF (Miltenyi, Biotec) for 7 days in 5% CO2 at 37 °C. On day 8, the medium was replaced with fresh RPMI-1640 and macrophages were plated with EOC cell lines.

Riillo C., Polerà N., Di Martino M.T., Juli G., Hokanson C.A., Odineca T., Signorelli S., Grillone K., Ascrizzi S., Mancuso A., Staropoli N., Caparello B., Cerra M., Nisticò G., Tagliaferri P., Crea R., Caracciolo D, & Tassone P. (2023). A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer. Journal of Translational Medicine, 21, 301.

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Antitumor Immune Response Analysis

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At day 11 after treatment initiation, B78 tumors (n=3) were harvested from mice treated with VO (cold NM600), anti-CTLA4 (IP, 200 ug day 4,7,10), 50 μCi ⁹⁰Y‐NM600 (day 1) or anti-CTLA4 + 50 μCi ⁹⁰Y‐NM600. Tumors were harvested and disassociated with DNAse and collagenase on a Miltenyi GentleMACS Octodissociator and then tumor infiltrating lymphocytes (TILs) were separated with magnetic bead separation using CD4/CD8 microbeads (Miltenyi Biotec). Isolated TILs were then spun down and diluted in media [RPMI 1640 containing 10% heat inactivated FBS and supplemented to a final concentration with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), 2-mercaptoethanol (50 μM), 150 Units/mL IL-2, and 1% ITS: insulin (1.7 μM), transferrin (68.8 μM) and sodium selenite (3.9 nM)], and placed in a 96 well plate previously coated with either 0, 1, or 5 ug/mL anti-CD3ε (Clone 145–2C11, BD). TILs were then allowed incubate for 48 hours in the presence of 5 ug/mL free anti-CD28 (clone 37.51, BD) in all wells. GolgiSTop (BD) was added 4 hours prior to collecting cells for staining. Cells were then prepped for flow cytometry as described above and differences in the Median Fluorescence Intensity (MFI) of IFN-γ in TILs was compared across treatment groups.

Patel R.B., Hernandez R., Carlson P., Grudinski J., Bates A.M., Jagodinsky J.C., Erbe A., Marsh I.R., Arthur I., Aluicio-Sarduy E., Sriramaneni R., Jin W.J., Massey C., Rakhmilevich A.L., Vail D., Engle J.W., Le T., Kim K., Bednarz B., Sondel P.M., Weichert J, & Morris Z.S. (2021). Low-dose targeted radionuclide therapy renders immunologically “cold” tumors responsive to immune checkpoint blockade. Science translational medicine, 13(602), eabb3631.

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PGE2 Modulation of T Cell PD-1 Expression

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Spleen was harvested from BALB/c mice. Single cell suspensions were prepared. CD4⁺ and CD8⁺ T cells were isolated by CD4/CD8 MicroBeads (Miltenyi Biotec). Cells were cultured in RPMI-1640 medium and stimulated by anti-CD3 (Invitrogen) and anti-CD28 (Invitrogen) in the presence or absence of various concentrations of PGE₂ (TCI) (10nM, 100nM, 1000nM). Cells were stained for PD-1 for flow cytometry analysis after 24 h treatment.

Huang H., Huang Y., Chen Y., Luo Z., Zhang Z., Sun R., Wan Z., Sun J., Lu B., Zhang L., Hu J, & Li S. (2021). A novel immunochemotherapy based on targeting of cyclooxygenase and induction of immunogenic cell death. Biomaterials, 270, 120708.

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Cytotoxic Evaluation of pAXLxCD3ε in EOC

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Healthy donors derived PBMCs were labelled with Cell-Trace Violet (Thermo Fisher Scientific) viable marker, according to manufacturer instructions. Labeled PBMCs were co-cultured with EOC cell line at different E:T ratio, in the presence of increasing concentrations of pAXLxCD3ε or vehicle for 48–72 h at 37 °C and 5% CO₂, and then stained with 7-AAD (BD Biosciences). Cytotoxicity was detected by flow cytometry (Attune NxT Flow cytometer, Thermo Fisher Scientific) as 7-AAD⁺/ Cell Trace Violet⁻ cells (%). In the cytotoxicity experiment with T cell depletion, immunomagnetic cell sorting using CD4, CD8 microbeads (MACS Miltenyi Biotec) was performed. In the cytotoxicity experiment with Fc blocking, Fc receptor binding inhibitor antibody (Invitrogen) was added to cell co-culture according to manufacturer instructions.
For 3D re-directed T cell cytotoxicity assay, after EOC spheroids formation 1 × 10⁶ Cell Trace Violet PBMCs were added to each well. Cells were treated with increasing concentration of pAXLxCD3ε or vehicle. After 72 h, spheroids were monitored by microscope scoring. After treatment spheroids were pooled and dissociated with trypsin–EDTA (0.05%). Cells were labelled with 7AAD viable marker for 15 min at room temperature. Finally, cells were analyzed by flow cytometer.

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