Anti cd8a microbeads

Manufactured by Miltenyi Biotec

Sourced in United States

Anti-CD8a microbeads are magnetic beads coated with antibodies specific for the CD8a molecule. They can be used for the isolation or enrichment of CD8+ T cells from various sample types.

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Lab products found in correlation

Rmil 27, by Thermo Fisher Scientific (2 mentions) Anti cd28, by Thermo Fisher Scientific (2 mentions) Anti cd44 antibody, by BioLegend (2 mentions) Anti cd3, by Thermo Fisher Scientific (2 mentions) Facsaria cell sorter, by BD (2 mentions) Anti cd62l, by BioLegend (2 mentions) Anti cd4 microbeads, by Miltenyi Biotec (2 mentions) Anti cd45, by Miltenyi Biotec (1 mentions) Multimacs system, by Miltenyi Biotec (1 mentions) Multimacs, by Miltenyi Biotec (1 mentions) Anti cd49b, by BioLegend (1 mentions) Macs ld column, by Miltenyi Biotec (1 mentions) Anti cd3ε, by BioLegend (1 mentions) Anti cd19, by BioLegend (1 mentions) Anti ly6g, by BioLegend (1 mentions) Anti biotin microbead, by Miltenyi Biotec (1 mentions) Macs ls column, by Miltenyi Biotec (1 mentions) Intracellular fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Ack lysis buffer, by Thermo Fisher Scientific (1 mentions) Facsaria, by BD (1 mentions)

9 protocols using anti cd8a microbeads

Isolation of Tumor-Infiltrating Lymphocytes

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Tumours were harvested at days 8–21 and digested for 40 min at 37 °C according to Tumour Dissociation Kit protocol (Miltenyi Biotec 130–096–730). Tumours were crushed on 100 μm cell strainers and washed twice with PBS 2% FCS. Single-cell suspensions were enriched for CD45⁺ or CD8⁺ cells using the MultiMACS system (Miltenyi Biotec). In brief, cells from tumour tissues were labelled with anti-CD45 or anti-CD8a microbeads (Miltenyi Biotec 130–052–301 or 130–117–044, respectively), and then purified using the POSSEL program on MultiMACS. The positive fraction was recovered for TIL analysis by flow cytometry or ex vivo assays.

Leclerc M., Voilin E., Gros G., Corgnac S., de Montpréville V., Validire P., Bismuth G, & Mami-Chouaib F. (2019). Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1. Nature Communications, 10, 3345.

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Mouse Infection Protocols for Viral Studies

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Mice were age- and sex-matched for each experiment and used at 8–10 weeks of age. Mice were infected as previously described for i.n. and s.s. infections [5 (link)]. For s.s. infections, fur was trimmed with clippers, then a thin layer of Vaseline was applied over the trimmed region and the remaining fur was shaved over with a double-edge razor blade one day before infection. Mice infected by i.p. or s.c. administration were injected with a volume of 100μL or 200μL of virus inoculum per mouse, respectively. For co-infection experiments, splenocytes isolated from B6 mice were infected at an MOI of 5, harvested 1 hpi, and washed three times with PBS. 1 x 10⁵ infected cells in 200 μL of PBS were transferred intravenously into naïve B6 mice. For the CPXV SDE protection experiment, CD8⁺ T cells from splenocytes of B6 mice that had been infected 7 days earlier with WT CPXV or MCMV were isolated by positive selection using anti CD8a MicroBeads (Miltenyi Biotec). 3 x 10⁶ CD8⁺ T cells were transferred intravenously into naïve B6 mice. Mice were infected approximately 24 h after transfer.

Lauron E.J., Yang L., Elliott J.I., Gainey M.D., Fremont D.H, & Yokoyama W.M. (2018). Cross-priming induces immunodomination in the presence of viral MHC class I inhibition. PLoS Pathogens, 14(2), e1006883.

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Isolation and Activation of Naive T Cells

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CD4⁺ and CD8⁺ T cells were purified from spleen and lymph nodes using anti-CD4 microbeads and anti-CD8a microbeads (Miltenyi Biotech) then stained in PBS with 0.5% BSA for 15 min on ice with anti-CD4, anti-CD8, anti-CD62L, and anti-CD44 antibodies (all from Biolegend, CA). Naïve CD4⁺ or CD8⁺ CD62L^highCD44^low T cells were sorted using the BD FACSAria cell sorter. Sorted cells were activated with plate bound anti-CD3 (2μg/ml for CD4 and 1μg/ml for CD8) and anti-CD28 (2μg/ml) in the presence of rmIL-27 (25ng/ml) (eBioscience). Cells were harvested at various time points for RNA, intracellular cytokine staining, and flow cytometry.

Chihara N., Madi A., Kondo T., Zhang H., Acharya N., Singer M., Nyman J., Marjanovic N.D., Kowalczyk M.S., Wang C., Kurtulus S., Law T., Etminan Y., Nevin J., Buckley C.D., Burkett P.R., Buenrostro J.D., Rozenblatt-Rosen O., Anderson A.C., Regev A, & Kuchroo V.K. (2018). Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature, 558(7710), 454-459.

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Isolation and Activation of Naive T Cells

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CD8+ T Cell Activation Assay

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CD8⁺ T cells were isolated from splenocytes of OT-I mice on C57BL/6 background using anti-CD8a microbeads (Miltenyi Biotec), and then fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE). Labeled CD8⁺ T cells and OVA-I peptide were then cocultured with matured DCs or VV-activated γδT cells at 1:1 ratio in 96 well plates. The cells were incubated at 37˚C for 72 hours, and then assessed via flow cytometry.

Dai R., Huang X, & Yang Y. (2021). γδT Cells Are Required for CD8+ T Cell Response to Vaccinia Viral Infection. Frontiers in Immunology, 12, 727046.

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Sorting Murine Bone Marrow Progenitor Cells

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For sorting BMPs, BM cells of naïve mice were labeled with biotinylated anti-CD3ε, anti-CD19, anti-CD49b, and anti-Ly6G (all from Biolegend) followed by anti-biotin microbeads (Miltenyi Biotech) and isolated using a MACS LD column (Miltenyi Biotech). The collected cells were further sorted into each BMP subset: total BMPs (lin⁻c-kit⁺CD11b⁻Ly6C⁻), cMoPs (lin⁻c-kit⁺CD115⁺CD135⁻CD11b⁻Ly6C⁺), MDPs (lin⁻c-kit⁺CD115⁺CD135⁺CD11b⁻Ly6C⁻), CD135⁺ BMPs (lin⁻c-kit⁺CD115⁻CD135⁺CD11b⁻Ly6C⁻), and CD115⁻CD135⁻ BMPs (lin⁻c-kit⁺CD115⁻CD135⁻CD11b⁻Ly6C⁻).
To isolate cDCs and moDCs from infected mice, Lin⁻ cells of infected splenocytes (day 4 or 8 p.i.) were isolated using a MACS LD column and further sorted to each subset (cDCs as lin⁻IA/IE⁺CD11c⁺CD11b⁻CCR2⁻Ly6C⁻ and moDCs as lin⁻IA/IE⁺CD11c^−/intCD11b⁺CCR2⁺Ly6C⁺).
CD45.1⁺ P14 cells used in the in vitro and in vivo experiments were enriched using anti-CD8a microbeads and a MACS LS column (Miltenyi Biotech) and further purified to CD45.1⁺CD8⁺ cells by cell sorting.
Cell sorting was conducted using a FACS Aria II or FACS Aria III. The purities of all sorted populations were >95%.

Shin K.S., Jeon I., Kim B.S., Kim I.K., Park Y.J., Koh C.H., Song B., Lee J.M., Lim J., Bae E.A., Seo H., Ban Y.H., Ha S.J, & Kang C.Y. (2019). Monocyte-Derived Dendritic Cells Dictate the Memory Differentiation of CD8+ T Cells During Acute Infection. Frontiers in Immunology, 10, 1887.

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Adoptive Transfer of Antigen-Specific CD8 T Cells

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Donor OT-1 CD8 T cells from wild-type CD45.1/2⁺ and Bach2^12del CD45.2⁺ donor mice were isolated by mashing spleens through a 70 μm strainer, followed by red blood cell lysis with ACK lysis buffer (Gibco) for 1 min and positive selection with anti-CD8a microbeads (Miltenyi 130-117-044). Cells were stained in 200 μl total volume for 20 min at 4 °C before sorting naïve OT-1 cells (live CD44⁻ CD62L⁺ CD8a⁺ TCR Vα2⁺ TCR Vβ5⁺, catalog number and dilution info in Supplementary Table 19). 5000 WT and 5000 Bach2^12del cells were co-transferred into 10 CD45.1 recipient mice by retroorbital injection, and recipient animals were intranasally infected the following day with 10⁴ PFU of VSV-OVA. After 7 days, spleens were collected from infected mice and processed as described above. Cells were stained for surface antigens (KLRG1, CD127, CD8A, CD45.1, CD45.2, CD62L, CD44, CX3CR1, CXCR3, info in Supplementary Table 19, see Supplementary Fig. 9 for gating strategy) for 20 min at 4 °C, fixed with Intracellular Fixation & Permeabilization Buffer (eBioscience), and stained for intracellular antigens (Tbet, Eomes, info in Supplementary Table 19) for 45 min at RT. Cells were analyzed on a Cytek Aurora and counting beads (Polysciences 18328–5) were used to enumerate cells and gated using FlowJo (10.8.1). Two independent experiments were conducted.

Mouri K., Guo M.H., de Boer C.G., Lissner M.M., Harten I.A., Newby G.A., DeBerg H.A., Platt W.F., Gentili M., Liu D.R., Campbell D.J., Hacohen N., Tewhey R, & Ray J.P. (2022). Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells. Nature genetics, 54(5), 603-612.

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Isolating and Stimulating CD8+ T Cells

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Parent P815 cells not expressing H-2^b MHC class I molecules and P815 cells stably expressing H-2D^b or H-2K^b MHC class I molecules were a gift from Dr. John Harty (University of Iowa). P815 cell lines were pulsed with 1 μM peptide at 37°C for 1 hour and washed twice. CD8⁺ T cells were isolated from the lungs using anti-CD8a microbeads (Miltenyi Biotec, Auburn, CA) and pre-sorted via positive selection on a MidiMACS separator (Miltenyi Biotec). Positively selected cells were stained extracellularly with a monoclonal antibody specific to CD8 (clone 53–6.7) and purified populations of CD8⁺ T cells were sorted using a FACSAria (BD Biosciences). Sort-purified CD8⁺ T cells were stimulated with either peptide-pulsed P815 cells or unpulsed P815 cells at a 2:1 effector-to-stimulator ratio for 5 hrs at 37°C in the presence of 10 μg/ml BFA in 10% FCS-supplemented RPMI.

Schmidt M.E, & Varga S.M. (2019). Identification of novel respiratory syncytial virus CD4+ and CD8+ T cell epitopes in C57BL/6 mice. ImmunoHorizons, 3(1), 1-12.

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Cytotoxic T Cell Quantification Assay

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For measurement of tumor-specific cytotoxic T cells, splenocytes and tumor-infiltrating lymphocytes (TILs) from each group were isolated 3 days after the final treatments. Cells were labeled with anti-CD8a microbeads (Miltenyi Biotec, Auburn, CA, USA), then purified using MACS (Miltenyi Biotec). CD8+ T cells were enriched to more than 90% purity and purified CD8+ T cells were co-cultured with Renca tumor cells (10:1 ratio) in 96-well plates which are precoated with mouse IFN-ɣ (Mabtech, Inc., Cincinnati, OH, USA). Plates were incubated with 1 μg/mL of the biotinylated anti-mouse IFN-ɣ antibody R4-6A2-biotin for 2 h at room temperature then addition and incubation of streptavidin-ALP solution for 1 h at room temperature. Finally, spots were analyzed using ImageJ software after the addition of BCIP/NBT-plus substrate solution.

Park J.S., Lee M.E., Jang W.S., Kim J., Park S.M., Oh K., Lee N, & Ham W.S. (2022). Systemic Injection of Oncolytic Vaccinia Virus Suppresses Primary Tumor Growth and Lung Metastasis in Metastatic Renal Cell Carcinoma by Remodeling Tumor Microenvironment. Biomedicines, 10(1), 173.

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