To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro30 (link). Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).
Counting beads
Manufactured by Merck Group
Sourced in United States
Counting beads are small, spherical particles used in various laboratory applications, such as cell counting and analysis. They serve as a reference standard to quantify the number of cells or particles in a sample. Counting beads are available in different sizes, materials, and colors to suit different experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (2 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Golgistop, by BD (2 mentions)
Cd8 53 6, by Cytek Biosciences (2 mentions)
Klrg1 2f1, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Cd3 17a2, by BioLegend (2 mentions)
Cd45 30 f11, by BD (2 mentions)
Anti ifn γ xmg1, by BioLegend (2 mentions)
Cd44 im7, by Cytek Biosciences (2 mentions)
Live dead ghost dye, by Cytek Biosciences (2 mentions)
Cd4 rm4, by Cytek Biosciences (2 mentions)
Fortessa 1770 flow cytometer, by Merck Group (2 mentions)
Dextran t500, by Merck Group (1 mentions)
Anti cd66b antibodies, by BioLegend (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
5 protocols using counting beads
1
Ex Vivo T Cell Functionality Analysis
2
Neutrophil Isolation and Tumor Tissue Analysis
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After ethic approval was obtained from NRES Committee London - Camden and Islington (reference 15/LO/0933), whole human blood was kindly given by our colleagues. Neutrophils were isolated by density gradient separation method using 3% Dextran T500 (Sigma-Aldrich, Dorset, UK). The purity and cell count of neutrophils were assessed by flow cytometry analysis using anti-CD45 and anti-CD66b antibodies (Biolegend, San Diego, CA) and counting beads (Sigma-Aldrich) respectively. Primary tumor tissues and normal tissues from cancer patients were kindly given by our colleagues from Henan Provincial Hospital, Zhengzhou, Henan, China (Ethics approval no. 201944). To minimize individual variation, all tumor tissue and its NAT were harvested from the same patient and analyzed for comparison. All tissue specimens were formalin-fixed and paraffin-embedded when received. Specimens were stored at -20°C for future use.
3
Cell Viability Assay with Trypan Blue
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During compound treatment over 6 days, cell counts for titration experiments and proliferation curves were determined by staining with Trypan blue (Gibco, Thermo Fisher Scientific) using the Neubauer counting chamber. Further, cell counts were analyzed via flow cytometry by staining dead cells with propidium iodide (PI, BD Biosciences, San Jose, CA, USA) and counting beads (Sigma-Aldrich) as previously described [6 (link)].
4
Ex Vivo T Cell Functionality Analysis
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To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro30 (link). Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).
5
Xenogeneic GVHD Model in NOD/Scid/γc−/− Mice
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Example 14
A published xenogeneic GVHD model was used (Hippen et al., Sci. Transl. Med., 2011, 3(83):83ra41). Briefly, NOD/Scid/γc−/− mice between 8-12 weeks old were housed in a pathogen-free facility in micro-isolator cages. On day 0, mice were irradiated with 50 cGy. Human PBMNCs (15×106) were injected with or without expanded tTregs (15×106). To document PBMC-associated peripheral T-cell expansion, animals were bled (10-40 μL), red blood cells lysed, and tTreg and PBMC subsets were enumerated by flow cytometry by staining with mAbs to human CD4, Foxp3, CD8, CD45, and HLA-A2 (to differentiate between PBMC and tTreg) and acquired with a known number of counting beads (Sigma-Aldrich). Mice were assessed for signs of GVHD daily, weighed thrice weekly, and human cells in blood quantitated by flow cytometry on the specified dates.
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