Mouse cd8 isolation kit

Manufactured by STEMCELL

Sourced in Canada

The Mouse CD8 Isolation Kit is a laboratory tool designed to isolate CD8+ T cells from mouse samples. It utilizes magnetic beads coated with antibodies specific to the CD8 surface marker to selectively bind and separate CD8+ cells from the sample. The isolated cells can then be used for further downstream applications.

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Lab products found in correlation

Live dead cell mediated cytotoxicity kit, by Thermo Fisher Scientific (1 mentions) Cyto tox 96 non radioactive cytotoxicity assay kit, by Beyotime (1 mentions) Anti cd3, by BioLegend (1 mentions) Anti cd28, by BioLegend (1 mentions) Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Sds polyacrylamide gel, by Bio-Rad (1 mentions) Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

Close spelling variants of this product

EasySep™ Mouse CD8+ or CD4+ T Cell Isolation Kits Isolation kits

5 protocols using mouse cd8 isolation kit

Quantifying CD8+ T Cell Cytotoxicity

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OT-1 CD8+ T cells were isolated from splenocytes of 6–8-week-old female OT-1 mice using mouse CD8 isolation kit (StemCell). Cells were cultured and expanded using immobilized anti-CD3 (10 μg ml⁻¹) and soluble anti-CD28 (2 μg ml⁻¹) for three days. The flow cytometry-based cell-mediated cytotoxicity assay was then performed using the LIVE/DEAD Cell-Mediated Cytotoxicity Kit (Thermo Scientific)^{19 (link)}. The B16-Ova cells were used as TCs and were labeled with DiO for 20 min at 37 °C. The activated OT-1 cells were then pre-treated with LB-100 for 4 h before mixing with the TC at the specified ET ratio. The cell mixture was then incubated for 3 h in the presence of PI and analyzed by FACS. The percentage (%) of effector cell induced cell death was calculated by subtracting the % of DiO+PI+ cells in the absence of effector cells from % of DiO+PI+ cells in each condition, thereby correcting for the rate of spontaneous target cell death.

Ho W.S., Wang H., Maggio D., Kovach J.S., Zhang Q., Song Q., Marincola F.M., Heiss J.D., Gilbert M.R., Lu R, & Zhuang Z. (2018). Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade. Nature Communications, 9, 2126.

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Exploring CD8+ T-Cell Cytotoxicity Against Cancer Cells

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Single-cell suspensions were extracted from the spleens of 6-week-old C57BL/6 mice to isolate CD8⁺ T-cells. CD8⁺ T cells were purified using a mouse CD8⁺ isolation kit (Stemcell, Canada). Then, the harvested CD8⁺ T cells were seeded at a density of 1×10⁵ in 96-well plates and supplemented with anti-CD28 (1 μg/mL; BioLegend), anti-CD3 (5 μg/mL; Biolegend), and IL-2 (40 U/mL) for 3 d to induce CD8⁺ T cell activation. SCC9 cells, given the different treatments (PBS, GDYO, cholesterol, GDYO + L + Cholesterol, GDYO + L), were co-cultured for 5 hours with activated T cells at various ratios of effector to target. Using a Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit (Beyotime, China), the supernatant from each well was used for a lactate dehydrogenase cytotoxicity assay to measure target cell mortality, according to the manufacturer’s recommended protocol. In other experiments, activated CD8⁺ T cells were added at the effector to target ratios of 10:1 for 5 h to quantify the death of cancer cells by flow cytometry, and then co-culture media were measured for the inflammatory mediators, including IFN-γ and TNF-α, using enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific, USA).

Zhang L., Pan K., Huang S., Zhang X., Zhu X., He Y., Chen X., Tang Y., Yuan L, & Yu D. (2023). Graphdiyne Oxide-Mediated Photodynamic Therapy Boosts Enhancive T-Cell Immune Responses by Increasing Cellular Stiffness. International Journal of Nanomedicine, 18, 797-812.

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Generating Effector OT-I CD8+ T Cells

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Naïve OT-I CD8⁺ T cells and splenocytes were isolated from the spleen of OT-I mice. CD8⁺ T cells were isolated using a mouse CD8⁺ isolation kit (StemCell). T cells and splenocytes were cultured in RPMI 1640 (GlutaMAX) with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 50 μM β-ME, 10mM HEPES and 1×MEM NEAA, 100 U/ml penicillin and 100 mg/ml streptomycin (later referred to as complete T cell culture media). Cytokines in T cell culture media were added as indicated. To generate effector CD8⁺ T cells, OT-I splenocytes were cultured in complete T cell media containing 1 nM SIINFEKL (OVA_257–264) peptide for 2 days, followed by the addition of 100 IU/mL rhIL-2. Effector CD8⁺ T cells were ready for use after another 4 days in culture. All cells cultures were incubated at 37 °C under 5% CO₂.

Liu Z., Li J.P., Chen M., Wu M., Shi Y., Li W., Teijaro J.R, & Wu P. (2020). Detecting Tumor Antigen-specific T cells via Interaction Dependent Fucosyl-biotinylation. Cell, 183(4), 1117-1133.e19.

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CD8+ T Cell Isolation and Immunoblot Analysis

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All cells were lysed using RIPA (Thermo Fisher) buffer plus protease and phosphatase inhibitors (Thermo Fisher). Equal amounts of protein were separated on SDS polyacrylamide gels (BioRad) and transferred to PVDF membranes (Millipore). Antibodies used for immunoblot analyses were against AR (Abcam, ab108341) and β-actin (Abcam, ab8226). Secondary antibodies included anti-rabbit DyLight800 IgG (Cell Signaling, 5151) and anti-mouse DyLight680 IgG (Cell Signaling, 5470).
To collect enough protein lysate for immunoblot analyses, spleens from WT AR^fl/(fl) and E8iCre-AR^fl/(fl) mice were mechanically homogenized, incubated with a red cell lysis buffer (Biolend, 420302) and passed through 70 micron filters. CD8⁺ T cells were isolated using a mouse CD8 isolation kit (Stemcell, 19853) according to manufacturer’s recommendations. Splenocytes were then stimulated using plate-bound 5 μg/mL CD3 (BioLegend, 100359) and 2 μg/mL CD28 (BioLegend, 102121) plus 40 ng/mL IL2 (NIH) for 72 hours. Afterwards, cells were washed and expanded in the presence of 40 ng/mL IL2 for another 72 hours before they were pelleted and lysed for immunoblot analyses.

Kwon H., Schafer J.M., Song N.J., Kaneko S., Li A., Xiao T., Ma A., Allen C., Das K., Zhou L., Riesenberg B., Chang Y., Weltge P., Velegraki M., Oh D.Y., Fong L., Ma Q., Sundi D., Chung D., Li X, & Li Z. (2022). Androgen Conspires with the CD8+ T Cell Exhaustion Program and Contributes to Sex Bias in Cancer. Science immunology, 7(73), eabq2630.

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Generating Effector CD8+ T Cells from OT-I Mice

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Naive OT-I CD8+ T cells and splenocytes were isolated from the spleen of OT-I mice. CD8+ T cells were isolated using a mouse CD8+ isolation kit (StemCell). T cells and splenocytes were cultured in RPMI 1640 (GlutaMAX) with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 50 mM b-ME, 10 mM HEPES, and 1 3 MEM-NEAA, 100 U/ml penicillin and 100 mg/ml streptomycin (later referred to as complete T-cell culture media). Cytokines in T-cell culture media were added as indicated. To generate effector CD8+ T cells, OT-I splenocytes were cultured in complete T-cell media containing 1 nM SIINFEKL (OVA257-264) peptide followed by the addition of 100 IU/mL rhIL-2. Effector CD8+ T cells were ready for use after 2–4 days in co-culture. All cells cultures were incubated at 37 °C under 5% CO₂.

Saddawi-Konefka R., O’Farrell A., Faraji F., Clubb L., Allevato M.M., Jensen S.M., Yung B.S., Wang Z., Wu V.H., Anang N.A., Msari R.A., Schokrpur S., Pietryga I.F., Molinolo A.A., Mesirov J.P., Simon A.B., Fox B.A., Bui J.D., Sharabi A., Cohen E.E., Califano J.A, & Gutkind J.S. (2022). Lymphatic-preserving treatment sequencing with immune checkpoint inhibition unleashes cDC1-dependent antitumor immunity in HNSCC. Nature Communications, 13, 4298.

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