The following reagents were used for cell stimulation, differentiation or treatment:αCD3 (10 μg/mL; BioLegend, San Diego, CA, 300438), αCD8 (2 μg/mL; BioLegend, 302934), dipyridamole (10 μM, R&D, 0691), IFNγ (20 ng/mL; PeproTech, Rocky Hill, NJ, 300-02), IL-1β (10 ng/mL; PeproTech, 200-01B), IL-2 (10 ng/mL; PeproTech, 200-02), IL-4 (20-25 ng/mL; PeproTech, 200-04), IL-6 (10 ng/mL; PeproTech, 200-06), IL-12 (10 ng/mL; PeproTech, 200-12), IL-23 (10 ng/mL; PeproTech, 200-23), ionomycin (10 μg/mL; Sigma, I0634), LPS (20 ng/mL to 1 μg/mL, Invivogen, tlrl-3pelps), M-CSF (10-25 ng/mL; PeproTech, 300-25), NBMPR (1 μM; R&D, 2924), PHA (4 μg/mL; Sigma, L1686), PMA (1 μg/mL; Sigma, P1585), transforming growth factor β (1 ng/mL; Miltenyi, 130-095-067), TNF-α (50 ng/mL; PeproTech, 300-01A), tofacitinib citrate (Tofa, 10-500 nM; MedChemExpress, HY-40354A), SLC29A1/ENT1 antibody (LSBio, #LS-B3385), and pSTAT3 antibody (Cell Signaling Technology, Danvers, MA; 9145).
α cd8
Manufactured by BioLegend
Sourced in United States
α-CD8 is a monoclonal antibody that specifically binds to the CD8 protein, which is expressed on the surface of cytotoxic T cells and a subset of natural killer cells. The antibody can be used for the identification and characterization of CD8-positive cells in various applications, such as flow cytometry and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
α cd45, by BioLegend (2 mentions)
Tlrl 3pelps, by Thermo Fisher Scientific (1 mentions)
P stat3 antibody, by Cell Signaling Technology (1 mentions)
Il 23, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tofacitinib citrate, by MedChemExpress (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Propidium iodide, by BioLegend (1 mentions)
Tcrγδ, by BD (1 mentions)
α cd4, by BioLegend (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Negative selection, by Miltenyi Biotec (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
α cd25, by BioLegend (1 mentions)
5 protocols using α cd8
1
Stimulation and Differentiation of Immune Cells
2
Isolation and Analysis of Cytotoxic T Cells
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CD8+ T cells were enriched from PBMCs by negative selection using magnetic bead separation according to the manufacturer’s instructions (STEMCELL Technologies), added to uninfected or infected A549 cells at a ratio of 3:1, and incubated for 16 h in the presence of α-TNF (Beckman Coulter) and TAPI-O (EMD Millipore) to prevent TNF cleavage from the cell surface (Haney et al., 2011 (link)). The following day, cells were stained with: (a) α-CD4 and α-CD8 (BioLegend) and (b) α–TRAV1-2 and α–TCRγδ (Thermo Fisher Scientific). Dead cells were excluded from the analysis using propidium iodide (BioLegend). Viable CD4−TCRγδ−CD8+TRAV1-2+TNF+ cells were sorted at high purity using a FACSAria flow cytometer (BD) directly into RNAlater (Applied Biosystems). The corresponding nonfunctional populations were sorted in parallel for control purposes. In all cases, cell numbers were standardized (5,000 per aliquot) to minimize sampling bias.
3
Tetramer Competition Assay for TCR Quantification
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Spleens and tumour draining lymph nodes were pooled from Treg depleted or replete mice. CD8+ T cells were purified from disaggregated tissues using magnetic isolation by negative selection (Miltenyi). Purified CD8+ T cells were incubated with 50 nM of dasatinib (New England Biolabs) to prevent TcR internalisation before staining with α-CD8, α-TCR β-chain (H57-597; Biolegend), and 5 μg of PE-labelled GSW11-specific tetramers. After two washes, cells were incubated with bleached tetramer at varying ratios of the initial PE-labelled tetramers: 2.5, 5, 10 or 20 μg per test. Bleached tetramers were tested for no/minimal PE-fluorescence before use. The β-chain TCR staining was included to confirm the decreasing levels of PE staining was due to the fluorescently labelled tetramer being out-competed and not due to TcR internalisation.
4
Comprehensive Tumor Immune Profiling
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Chopped tumor tissues were enzymatically digested using the dissociation buffer supplemented with 1 mg/ml Collagenase B (11088807001, Roche) and 1 mg/ml Hyaluronidase (H3506, Sigma-Aldrich) for 1 h at 37 °C. The prepared single-cell suspensions were filtered through 40-μm nylon meshes (352340, Corning) and centrifuged at 400 g for 5 min. Then, the centrifuged cells were treated with red blood cell lysis buffer (C3702, Beyotime). Subsequently, the cells were dyed with Fixable Viability Stain 780 (565388, BD), and Fc receptors were blocked with Ultra-LEAF™ Purified anti-mouse CD16/32 (101320, BioLegend). Cells were then fluorescently stained with the following detection antibodies: α-CD45 (103132, BioLegend), α-CD3 (100206, BioLegend), α-CD8 (100706, BioLegend), α-Ki67 (151215, BioLegend), α-CD69 (104536, BioLegend), α-CD25 (102012, BioLegend), α-CD107a (121629, BioLegend), α-Granzyme-B (372214, BioLegend), α-IFN-γ (505838, BioLegend). Brilliant Stain Buffer (563794, BD Biosciences) and True-Nuclear™ Transcription Factor Buffer Set (424401, BioLegend) were used in this assay. Flow cytometry was performed using Beckman CytoFLEX LX, and the data were analyzed by FlowJo_V10.
5
Generation of Bone Marrow Chimeras
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BM chimeras were generated by irradiating B6 (CD45.1) recipient mice twice with 550 Rads, 3 h apart. BM from hind legs of donor mice was flushed and T cells depleted with anti‐CD4 (clone RL172), anti‐CD8 (clone 3.168), anti‐Thy1 (clone Jlj) and rabbit complement. Incubation of BM with antibodies was performed for 30 min on ice followed by cell resuspension in 1 mL of complement for 20 min at 37°C. About 5 × 106 T‐cell–depleted BM cells were injected intravenously into recipient mice; 24 h after irradiation, recipient mice were injected intraperitoneally with 100 μL of α‐CD4 and α‐CD8 T‐cell monoclonal antibodies (clone RL172 and clone 3.168, respectively) to eliminate radioresistant T cells. Submandibular bleeds were performed > 8 weeks after irradiation to determine BM reconstitution by staining blood with a cocktail of α‐CD45.1 (eBioscience, Fischer Scientific, Waltham, MA, USA), α‐CD45.2 (BioLegend, San Diego, CA, USA), anti‐CD4 (BioLegend), α‐CD8 (BioLegend) and α‐CD62L (BioLegend). Live cells were discriminated with a fixable LIVE/DEAD stain (Life Technologies, Carlsbad, CA, USA).
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