Cd4 and cd8 t cell isolation kits

Manufactured by Miltenyi Biotec

The CD4+ and CD8+ T cell isolation kits from Miltenyi Biotec are designed to isolate T cell subsets from a variety of biological samples. The kits utilize magnetic bead-based technology to selectively enrich for CD4+ or CD8+ T cells, enabling the user to obtain highly purified populations of these cell types.

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Lab products found in correlation

Facs influx flow cytometer, by BD (1 mentions) Ficoll, by GE Healthcare (1 mentions) Cd14 microbead, by Miltenyi Biotec (1 mentions) Lysis buffer, by BD (1 mentions) Macs buffer, by Miltenyi Biotec (1 mentions) Automacs pro separator, by Miltenyi Biotec (1 mentions)

3 protocols using cd4 and cd8 t cell isolation kits

Evaluating T Cell Activation by APC Subsets

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CD4⁺ and CD8⁺ T cells from spleens of OT-2 or OT-1 transgenic mice were purified using CD4⁺ and CD8⁺ T cell isolation kits (Miltenyi Biotech.) following the manufacturer's instructions, respectively. To evaluate the T cell activation capacity of different DCs subsets, CD8⁺ DCs, CD8⁻ DCs, and pDCs from splenocytes of naïve C57BL/6 mice were isolated by FACS (BD FACS Influx Flow Cytometer) and used as APCs. Graded numbers of APCs in the presence of rOVA or rOVA-FLIPr (10 μg/ml) were cocultured with purified OT-2 CD4⁺ and OT-1 CD8⁺ T cells (5 × 10⁵ T cells/well) for 40 and 24 h, respectively. For some experiments, single suspension lymphocytes were prepared from lymph nodes which derived from mice injected with rOVA or rOVA-FLIPr. The CD11c⁺ and CD11c⁻ cells isolated by pan DC isolation kits or CD8⁺ DCs and CD8⁻ DCs isolated by FACS (BD FACS Influx Flow Cytometer) were used as APCs. Purified OT-2 CD4⁺ and OT-1 CD8⁺ T cells (5 × 10⁵ T cells/well) were cocultured with APCs without further addition of rOVA or rOVA-FLIPr for 40 and 24 h, respectively. Levels of IL-2 and IFN-γ in the supernatants were determined by ELISA.

Chiang C.Y., Wu C.C., Chen Y.J., Liu S.J., Leng C.H, & Chen H.W. (2019). Delivery of Antigen to CD8+ Dendritic Cells by Fusing Antigen With Formyl Peptide Receptor-Like 1 Inhibitor Protein Induces Antitumor Immunity. Frontiers in Immunology, 10, 1839.

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Isolation of Immune Cell Subsets

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Blood samples were obtained from healthy volunteers with authorization from the National Research Ethics Services, Westminster (09/H0715/30), and with informed written consent. Neutrophils were isolated directly from peripheral blood using the Neutrophil Isolation Kit/MACSexpress (Miltenyi Biotec, Bergisch Gladbach, Germany). Peripheral blood mononuclear cells were collected after centrifugation of whole blood (diluted 1:1 in phosphate buffered saline) over Ficoll (GE Healthcare, Little Chalfont, UK). CD14⁺ cells and CD4⁺ and CD8⁺ T cells were isolated from peripheral blood mononuclear cells using CD14 Microbeads and CD4⁺ and CD8⁺ T-cell isolation kits, respectively (Miltenyi Biotec) following the manufacturer’s instructions. Purity of CD8⁺ and CD4⁺ T cells was analyzed by flow cytometry and was typically greater than 95%. DCs were generated using our established laboratory protocols. Briefly, CD14⁺ cells were cultured for 6 days in RPMI supplemented with 100 ng/ml GM-CSF and 25 ng/ml IL-4, which typically yields greater than 95% CD11c⁺ cells with an immature phenotype (Burns et al., 2004 (link); Metelo et al., 2011 (link)) and a characteristic dendritic appearance after adhesion to substrate (Figure 5a).

Nowak K., Linzner D., Thrasher A.J., Lambert P.F., Di W.L, & Burns S.O. (2017). Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment. The Journal of investigative dermatology, 137(10), 2120-2130.

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Adoptive Transfer of CD4+ and CD8+ T cells

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CD4⁺ and CD8⁺ T cells were purified from the spleens of OTII and OTI mice, respectively, by magnetic labeling in an autoMACs® Pro Separator (Miltenyi Biotec, San Diego, CA). Briefly, spleens were disaggregated and filtered into single cell suspensions, and RBCs were removed by incubating for 5 min in 1X lysis buffer (BD Biosciences). The splenocytes were then washed once in RPMI media and once in MACs buffer (Miltenyi Biotec) and resuspended at 2.5 x 10⁸ cells per mL. The cells were then subject to magnetic labeling and T cell isolation using CD4⁺ and CD8⁺ T cell isolation kits (Miltenyi Biotec), according to the manufacturer’s instructions. The purified CD4⁺ and CD8⁺ T cells were then mixed at a 2:1 ratio in PBS at a total cell concentration of 3 × 10⁷ per mL, and 100 µL of the appropriate cell mixture was injected intravenously in the tail veins of wildtype C57BL/6 mice.

Riccione K.A., He L.Z., Fecci P.E., Norberg P.K., Suryadevara C.M., Swartz A., Healy P., Reap E., Keler T., Li Q.J., Congdon K.L., Sanchez-Perez L, & Sampson J.H. (2018). CD27 stimulation unveils the efficacy of linked class I/II peptide vaccines in poorly immunogenic tumors by orchestrating a coordinated CD4/CD8 T cell response. Oncoimmunology, 7(12), e1502904.

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