Goat anti-hamster IgG (cat#127-005-160, Jackson ImmunoReserach) was pre-coated overnight at 4 °C in 96-well plates (5 µg/ml), and after blocking, anti-CD3 mAb (145-2C11; cat# MO3PU(V100), Immunostep) (1 µg/ml) was added and incubated at 37 °C for 1 h. Mouse CD8a+ T cells were purified from the spleens of C57BL/6 mice using the EasySep™ Mouse CD8a+T Cell Isolation Kit (Stem Cell Technologies). Then purified mouse CD8a+ T cells (2.5 × 105/well) in complete RPMI+50 µM 2-mercaptoethanol, and purified antibodies at 6.67 nM were added. As a control, purified mouse CD8a+ T cells were cultured alone with the immobilized anti-CD3 mAb (1 µg/ml). After 48 h, cell proliferation was assessed with the CellTiter-Glo luminescent assay (Promega) using a Tecan Infinite F200 plate-reading luminometer, and supernatants were collected and assayed for IFN-γ secretion by ELISA (Diaclone). For viability assays, cells were collected after 72 h, incubated with FITC-Annexin V (Immunostep) and 7-AAD (BD Biosciences), and analyzed with a MACSQuant Analyzer 10. Results are expressed as a mean ± SD from one of at least three separate experiments.
Macsquant analyzer 10
Manufactured by BD
Sourced in Germany
The MACSQuant Analyzer 10 is a compact and flexible flow cytometer designed for multi-parameter cell analysis. It features high-performance optics, sensitive detectors, and intuitive software to deliver accurate and reliable results. The instrument is capable of analyzing a wide range of sample types, including cells, beads, and particles, and can be configured with a variety of fluorescent parameters to meet the needs of diverse research applications.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Immunostep (1 mentions)
Celltiter glo luminescent assay, by Promega (1 mentions)
7 aad, by BD (1 mentions)
Pe anti mouse cd138 syndecan 1, by BioLegend (1 mentions)
Pi rnase buffer, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Dhr123, by Thermo Fisher Scientific (1 mentions)
Liberase tl, by Merck Group (1 mentions)
Anti cd16 32, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Tnf α, by BioLegend (1 mentions)
Mhcii, by BioLegend (1 mentions)
Cd11b, by BioLegend (1 mentions)
4 protocols using macsquant analyzer 10
1
Assessment of Murine CD8+ T Cell Activation
2
Isolation and Analysis of Mouse Immune Cells
3
Cell Cycle Analysis with NTX
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T24, T24/DOX, and T24/CIS (2 × 105 /well) were seeded in 6-well plates. After 24 h incubation with NTX (0, 10, 20, or 40 µM), cells were rinsed twice with PBS and fixed in precooling 70% ethanol for over 18 h at 4℃. Subsequently, they were rinsed twice with PBS and stain buffer (BD Biosciences, #554656). Following by 15 min staining in PI/RNase buffer (BD Biosciences, #550825), the analysis was performed by MACSQuant Analyzer 10 and MACSQuantifyTM Software 2.6.
4
Immune Cell Isolation and Characterization
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Organs were incubated in RPMI medium containing liberase TL (50 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and DNase I (50 mg/mL, Sigma-AldrichSt. Louis, MO, USA) for 90 min at 37 °C, passed through a 100 µm cell strainer, and centrifuged for 5 min, 4 °C, 500× g. Erythrocytes were lysed by ammonium chloride buffer (5 min, RT). Cells were incubated for 10 min at RT in 50 µL Fc-blocking buffer, containing anti-CD16/32 (0.5 µg/100 µL) (Thermo Scientific, Waltham, MA, USA), 1% rat serum, and 1% Syrian hamster serum, followed by indicated antibody (CD3, CD4, CD8e, CD11c, CD49b, CD69 all 1:200; CD11b, Ly6G both 1:400; IFN-g, MHC-II, MPO, TNF-a all 1:400) (Biolegend, San Diego, CA, USA) incubation together with DHR123 (50 µM) (Thermo Scientific, Waltham, MA, USA) for ROS and/or 7-AAD (1 µg/mL) (Thermo Scientific, Waltham, MA, USA) for necrosis assessment for 15 min on ice. For intracellular cytokine stainings, cells were treated with 5 µg/mL Brefeldin A in RPMI buffer in an anti-CD3/CD28-coated (5 µg/mL each) (Biolegend, San Diego, CA, USA) plate for 4,5 h at 37 °C with 5% CO2 followed by antibody incubation. Cells were analyzed with a MACS Quant Analyzer 10 (BD, Heidelberg, Germany).
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