Negative magnetic selection kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The Negative Magnetic Selection Kit is a lab equipment product designed for the isolation of specific cell types from complex cell samples. It utilizes magnetic beads coated with antibodies to negatively select the desired cells by removing unwanted cells from the sample.

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Negative selection (51 citations) Negative selection kit (47 citations) Negative magnetic selection (33 citations) Magnetic bead negative selection kit (11 citations) Negative selection magnetic bead isolation kit (2 citations) Magnetic bead-based negative selection kit (2 citations) CD8 magnetic bead negative selection kit (2 citations) Negative selection magnetic microbeads kit (2 citations)

3 protocols using negative magnetic selection kit

Isolation of Monocytes and CD8+ T Cells from PBMCs

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PBMCs were suspended in PBS supplemented with 0.5% BSA and 2 mm of EDTA (1 × 10⁷ cells/0.1 ml) and incubated with microbeads coated with mAb CD14 (Miltenyi Biotech, Bergisch Gladbach, Germany) to obtain an enriched monocytes fraction by a positive selection system. The purity percentage of CD14⁺ cells was routinely 90%–95%, as measured by flow cytometry.
The negative fraction of CD14 was suspended in PBS supplemented with 0.5% BSA, and 2 mm of EDTA (1 × 10⁷ cells/0.1 ml) was incubated with microbeads coated with an mAb cocktail (CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a) by a negative magnetic selection kit according to manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity percentage of CD8⁺ T cells was habitually 93%–97%, as determined by flow cytometry.

Chávez‐Galán L., Illescas‐Eugenio J., Alvarez‐Sekely M., Baez‐Saldaña R., Chávez R, & Lascurain R. (2019). Tuberculosis patients display a high proportion of CD8+ T cells with a high cytotoxic potential. Microbiology and Immunology, 63(8), 316-327.

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PD-1 Modulation of T Cell Activation

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PD-1-overexpressing Jurkat cells as previously published (23 ) were stimulated with Dynabeads conjugated to α-CD3 (UCHT1), α-CD28 (28.2), and α-PD-1 (clone EH12 from Gordon Freeman) or control IgG at a 4:1 bead:cell ratio in the presence of 1 µM CDK4/6 inhibitor for 18h. For primary human T cells, normal donor human blood was obtained through DFCI IRB Protocol 04–430. PBMC were isolated using a Ficoll-Paque density gradient, and purified populations of CD4+ T lymphocytes were obtained through a negative magnetic selection kit according to manufacturer’s instructions (Miltenyi). Primary human CD4+ T cells were stimulated with Dynabeads conjugated to α-CD3 (UCHT1), α-CD28 (28.2), and recombinant hPD-L1-IgG fusion protein (Gordon Freeman) or control IgG at a 4:1 bead:cell ratio in the presence of 1 µM CDK4/6 inhibitor for 18h. IL-2 levels in the supernatant were analyzed by AlphaLISA (Perkin Elmers) according to the manufacturer’s protocol.

Deng J., Wang E.S., Jenkins R.W., Li S., Dries R., Yates K., Chhabra S., Huang W., Liu H., Aref A.R., Ivanova E., Paweletz C.P., Bowden M., Zhou C.W., Herter-Sprie G.S., Sorrentino J.A., Bisi J.E., Lizotte P.H., Merlino A.A., Quinn M.M., Bufe L.E., Yang A., Zhang Y., Zhang H., Gao P., Chen T., Cavanaugh M.E., Rode A.J., Haines E., Roberts P.J., Strum J.C., Richards W.G., Lorch J.H., Parangi S., Gunda V., Boland G.M., Bueno R., Palakurthi S., Freeman G.J., Ritz J., Haining W.N., Sharpless N.E., Arthanari H., Shapiro G.I., Barbie D.A., Gray N.S, & Wong K.K. (2017). CDK4/6 Inhibition Augments Anti-Tumor Immunity by Enhancing T Cell Activation. Cancer discovery, 8(2), 216-233.

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Isolation of Cytotoxic CD8+ T Cells

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Cytotoxic CD8⁺ T cells were isolated from PBMCs by a negative magnetic selection kit (Miltenyi Biotec). Briefly, PBMCs (1×10⁷ cells) were suspended in 40 µl phosphate-buffered saline (PBS: 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) supplemented with 0.5% BSA and 2 mM EDTA and incubated with biotin-antibodies to human leukocyte phenotype molecules for 10 min at 4°C, followed by a second incubation with magnetic microbeads coated with mouse Abs against biotin and human CD14 for an additional 15 min at 4°C. The purity percentage for magnetically unlabeled CD8⁺ T cells was always >95%, as determined by flow cytometry via incubation with PE-Cy5.5-anti-human CD3, PE-anti-CD4, and FITC-anti-CD8 mAbs for 30 min at 4°C. The magnetically labeled CD8⁻ T cells were used to identify HLA-A2 alleles.

ATZIN-MÉNDEZ J.A., LÓPEZ-GONZÁLEZ J.S., BÁEZ R., ARENAS-DEL ANGEL M.C., MONTAÑO L.F., SILVA-ADAYA D., LASCURAIN R, & GOROCICA P. (2015). Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-β2 microglobulin complexes. Oncology Reports, 35(1), 33-42.

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