Human cd4 beads

Manufactured by Miltenyi Biotec

Sourced in Germany

Human CD4 beads are a magnetic cell separation reagent used for the isolation and enrichment of human CD4+ T cells from a variety of cell sources, such as peripheral blood, leukapheresis samples, or other cell preparations. The beads are coated with antibodies specific to the CD4 surface antigen, allowing for the efficient and gentle isolation of CD4+ T cells.

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Lab products found in correlation

Amaxa nucleofector, by Lonza (4 mentions) Human t cell nucleofector kit, by Lonza (4 mentions) Human t cell culture medium, by Lonza (3 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Soluble anti cd28 monoclonal antibodies, by Thermo Fisher Scientific (1 mentions) Density gradient centrifugation, by GE Healthcare (1 mentions) Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions) Anti cd3 antibody, by Thermo Fisher Scientific (1 mentions) Soluble anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)

6 protocols using human cd4 beads

Isolation and Stimulation of CD4+ T Cells from aGVHD Patients

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CD4⁺ T cells were purified from 60 mL of venous peripheral blood from patients with aGVHD using human CD4 beads, according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated CD4⁺ T cells were cultured in human T cell culture medium (Lonza, Basel, Switzerland), and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
For CD4⁺ T cell stimulation in vitro, the cells were plated at a density of 1–5 × 10⁶/well on 24-well plates containing 2.5 mg/mL plate-bound anti-CD3 with 1 mg/mL soluble anti-CD28 monoclonal antibodies (eBioscience, San Diego, CA, USA).

Xu Y.J., Chen F.P., Chen Y., Fu B., Liu E.Y., Zou L, & Liu L.X. (2018). A Possible Reason to Induce Acute Graft-vs.-Host Disease After Hematopoietic Stem Cell Transplantation: Lack of Sirtuin-1 in CD4+ T Cells. Frontiers in Immunology, 9, 3078.

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Electrotransfection of CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient. CD4⁺ T cells were purified from PBMCs using human CD4 beads (Miltenyi, Bergisch Gladbach, Germany) and cultured in RPMI 1640 (Thermo Fisher Scientific, MA, USA) containing 10% FBS, and 1% penicillin/streptomycin. The isolated CD4⁺ T cells were electrotransfected at 2 × 10⁶/sample. Human T cell nucleofector Kit and Amaxa nucleofector (Lonza, MD, USA) were used to transfect the gene expression (pCMV6) and interference (pRS) plasmids into CD4⁺ T cells. Briefly, CD4⁺ T cells were mixed with the plasmid in 100 μl of human T cell nucleofector solution. The mixture was electrotransfected using nucleofector program V-024 in the Amaxa nucleofector. The transfected cells were cultured in RPMI 1640 complete medium for 48 h.

Zeng C., Cheng T.T., Ma X., Liu Y., Hua J., Chen X., Wang S.Y, & Xu Y.J. (2022). The absence of AhR in CD4+ T cells in patients with acute graft-versus-host disease may be related to insufficient CTCF expression. Clinical Epigenetics, 14, 109.

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Overexpression and Interference in CD4+ T Cells

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CD4⁺ T cells were extracted from 60 ml venous peripheral blood with human CD4 beads (Miltenyi, Bergisch Gladbach, Germany) as directed by the manufacturer and cultured in RPMI 1640 (Thermo Fisher Scientific, MA, United States) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The gene overexpression (pCMV6) and interference (pRS) plasmids were transfected into 2 × 10⁶ CD4⁺ T cells with Human T cell Nucleofector Kit and Amaxa Nucleofector (Lonza, Walkersville, MD, United States). Briefly, CD4⁺ T cells were isolated, resuspended in 100 μl human T cell Nucleofector solution, and mixed with the plasmid. The resulting mixture was electrotransfected with Nucleofector program V-024 on an Amaxa Nucleofector. The transfected cells were cultured in RPMI 1640 containing 10% FBS at 37°C with 5% CO₂ and collected 48 h later.

Hua J., Chen Y., Fu B., Chen X., Xu X.J., Yang S.H., Chen C, & Xu Y.J. (2020). Downregulation of p53 by Insufficient CTCF in CD4+ T Cells Is an Important Factor Inducing Acute Graft-Versus-Host Disease. Frontiers in Immunology, 11, 568637.

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Isolation of CD4+ T cells

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Venous peripheral blood was withdrawn from each healthy control and patient and preserved in heparin. Peripheral blood mononuclear cells were then separated by density gradient centrifugation (GE Healthcare). CD4⁺ T cells were isolated by positive selection using human CD4 beads, following the manufacturer’s protocol (Miltenyi). The purity of the CD4⁺ T cells was evaluated by flow cytometry and was generally higher than 95%.

Luo S., Zhang H., Xie Y., Huang J., Luo D, & Zhang Q. (2022). Decreased SUV39H1 at the promoter region leads to increased CREMα and accelerates autoimmune response in CD4+ T cells from patients with systemic lupus erythematosus. Clinical Epigenetics, 14, 181.

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Efficient CD4+ T Cell Transfection

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CD4⁺ T cells were extracted from 40 ml venous peripheral blood using human CD4 beads (Miltenyi, Bergisch Gladbach, Germany) and cultured in human T cell culture medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. CD4⁺ T cells were transfected using the human T cell nucleofector kit and Amaxa nucleofector (Lonza, Walkersville, MD, USA). Briefly, CD4⁺ T cells were collected and resuspended in 100 μl human T cell nucleofector solution, and then the cell suspension was mixed with plasmids. The mix was electrotransfected using the nucleofector program V-024 in the Amaxa nucleofector. The transfected cells were cultured in human T cell culture medium and harvested after 48 h. Jurkat cells were cultured in the RPMI 1640 media (Gibco, Rockville, MD, USA) containing 10% FBS and incubated at 37°C in 5% CO₂. The plasmids were transfected into Jurkat cells via electrotransfection as described above.

Xu X., Chen Y., Liu E., Fu B., Hua J., Chen X, & Xu Y. (2020). HMGB1 Recruits TET2/AID/TDG to Induce DNA Demethylation in STAT3 Promoter in CD4+ T Cells from aGVHD Patients. Journal of Immunology Research, 2020, 7165230.

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Activation and Transfection of CD4+ T Cells

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CD4⁺ T cells were purified from 60 ml of venous peripheral blood using human CD4 beads according to the protocols provided by the manufacturer (Miltenyi, Bergisch Gladbach, Germany) and cultured in human T cell culture medium (Lonza, Walkersville, MD, USA). Purified normal CD4⁺ T cells were cultured in 24-well plates (1 × 10⁶/ml) and stimulated with plate-bound anti-CD3 antibody (eBioscience, CA, USA), followed by the addition of soluble anti-CD28 antibody (eBioscience) and incubation at 37 °C for 24 h. CD4⁺ T cells were transfected with the pCMV6 gene expression plasmid or the pRS gene interference plasmid using a Human T cell Nucleofector Kit and Amaxa nucleofector (Lonza). Briefly, CD4⁺ T cells were harvested and resuspended in 100 µl of human T cell nucleofector solution and then mixed with plasmid. The mixture was then used to electrotransfect T cells using the nucleofector program V-024 in the Amaxa nucleofector. The transfected cells were cultured in human T cell culture medium and harvested after 48 h.

Ding S., Zhang Q., Luo S., Gao L., Huang J., Lu J., Chen J., Zeng Q., Guo A., Zeng J, & Lu Q. (2019). BCL-6 suppresses miR-142-3p/5p expression in SLE CD4+ T cells by modulating histone methylation and acetylation of the miR-142 promoter. Cellular and Molecular Immunology, 17(5), 474-482.

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