402 ml 020 cf

Manufactured by R&D Systems

The 402-ML-020/CF is a piece of laboratory equipment. It is designed for use in research and scientific applications. The core function of this product is to perform specific tasks in a controlled laboratory environment. No further details about the intended use or capabilities of this product can be provided in an unbiased and factual manner.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (2 mentions) Anti cd28 antibodies clone 37, by BioLegend (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Cd8α t cell isolation kit, by Miltenyi Biotec (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) L glutamine, by Merck Group (1 mentions) Macsquant analyzer 10, by Miltenyi Biotec (1 mentions) Precision counting beads, by BioLegend (1 mentions) Igg2b, by BioXCell (1 mentions) Be0249, by BioXCell (1 mentions) Transwell insert, by Corning (1 mentions)

3 protocols using 402 ml 020 cf

Activation and Culture of CD8+ T Cells

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For activated CD8⁺ T cells, T-25 or T-75 flasks were coated with 5 µg/mL anti-CD3ϵ antibodies (clone 145-2C11; 100340, BioLegend) in phosphate-buffered saline (PBS) for 16–24 hr at 4°C before seeding CD8⁺ T cells. Immediately after magnetic isolation, CD8⁺ T cells were resuspended in complete medium (RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 µM 2-mercaptoethanol and 10 mM HEPES) and seeded in the antibody-coated flask at 3–5×10⁵ cells/cm² together with soluble 1 µg/mL anti-CD28 antibodies (clone 37.51; 102116, BioLegend). Cells were cultured for 48 hr at 37°C in a humidified 5% CO₂ atmosphere. For naïve CD8⁺ T cells, magnetically isolated T cells were resuspended in the complete medium and were seeded in a 10-cm petri dish or T-75 flask at 1–2×10⁶ cells/cm² and cultured in the presence of 20 ng/mL recombinant mouse interleukin-7 (rmIL-7; 402-ML-020/CF, R&D Systems, Minneapolis, MN) for 24 hr at 37°C in a humidified 5% CO₂ atmosphere. In some experiments, up to 1×10⁷ naïve CD8⁺ T cells were labelled with 5 µM CellTrace™ Violet (C34571, Thermo Fisher) for 20 min at 37°C in 1 mL complete medium before culturing them with rmIL-7 prior to nucleofection.

Pfenninger P., Yerly L, & Abe J. (2022). Naïve Primary Mouse CD8+ T Cells Retain In Vivo Immune Responsiveness After Electroporation-Based CRISPR/Cas9 Genetic Engineering. Frontiers in Immunology, 13, 777113.

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Activated OT-1 T Cell Cytotoxicity Assay

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CD8+ T cells were isolated from OT-1 mouse splenocytes using CD8α+ T cell isolation Kit (130-104-075, Miltenyi Biotec). Isolated T cells were cultured in RPMI 1640, supplemented with 10% heat inactivated FBS, 20 mM HEPES (15630080, Gibco), 1mM sodium pyruvate (11360070, Gibco), 0.05mM 2-mercaptoethanol, 2 mM L-glutamine (G7513, Sigma), and 100 U/ml penicillin streptomycin. For activation, Dynabeads Mouse T-Activator CD3/CD28 (11456D, Thermo Fisher) were added to isolated T cells at a bead-to-cell ratio of 1:2. Recombinant mouse IL-2 (402-ML-020/CF, R&D systems) was added to a final concentration of 20 ng/ml on Day 1 after isolation. Medium supplemented with 20 ng/ml IL-2 was changed every 2 days. T cells were split every 2 days to keep an approximate cell density of 10^6 cells/ml. Activated OT-1 T cells were used for co-culture killing assay on Day 6–8 after isolation. For co-culture killing assays, mouse IFNγ (20 ng/ml) pre-stimulated tumor cells were pulsed with 0.02 nM of SIINFEKL peptide for 2 hours at 37 degrees. Activated OT-1 cells were counted and plated with washed tumor cells at indicated E:T ratios. After 24 hours of coculture, T cells were gently washed off using PBS. Surviving cells were harvested and stained with mouse CD45.2 antibodies to gate out residual T cells. Surviving tumor cells were counted using a MACSQuant Analyzer 10 (Miltenyi).

Zhou L., Mudianto T., Ma X., Riley R, & Uppaluri R. (2019). Targeting EZH2 enhances antigen presentation, antitumor immunity and circumvents anti-PD-1 resistance in head and neck cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(1), 290-300.

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Quantifying T Cell Activation and Migration

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T cell activation and migration assays were performed following the protocol by Albert et al. (23 (link)). Briefly, splenocytes from OT-1 transgenic mice were stimulated with 1 nM TCR-specific peptide ovalbumin 257–264 ( Sigma-Aldrich, S7951) for 1 hour. Seven days after culture in complete RPMI 1640 medium with 50 U/mL of recombinant IL-2 (R&D, 402-ML-020/CF), CD8⁺ T cells with high CXCR3 expression were used for T cell migration assay. To inhibit receptor binding of CXCR3, anti-mouse CXCR3 mAbs (10 μg/mL, BioXcell, BE0249) were applied to treat activated OT-1 cells for 1 hour at 37°C; IgG2b-pretreated (10 μg/mL, BioXcell, BP0090) cells were used as isotype control. Then, 0.1 × 10⁶ activated T cells were placed into 5 μm pore size polystyrene Transwell inserts (Corning, 3421) in serum-free RPMI 1640 and allowed to migrate for 1.5 hours at 37°C toward EO771-GFP/Bic cell conditioned medium. Cells that had migrated to the receipt chamber were collected and counted using Precision Counting Beads (BioLegend, 424902) by flow cytometry.

Wang J., Wang Q., Guan Y., Sun Y., Wang X., Lively K., Wang Y., Luo M., Kim J.A., Murphy E.A., Yao Y., Cai G, & Fan D. (2022). Breast cancer cell–derived microRNA-155 suppresses tumor progression via enhancing immune cell recruitment and antitumor function. The Journal of Clinical Investigation, 132(19), e157248.

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