Twelve-well non-tissue culture-treated plates (Corning #351143) were coated at 4 °C overnight with 1 µg/mL anti-CD3 antibody (BD biosciences 555329) and 1.5 µM human IgG Fc fragment (Jackson Immunoresearch #009-000-008) or 1.5 µM recombinant human Fc-tagged PD-L1 (rhPD-L1.Fc) (Sino Biological #10084-H02H) in 1.2 mL PBS per well. The plate was washed twice with 2 mL PBS and blocked for 1 h at room temperature with 1.5 mL human serum albumin 2.5% in phosphate-buffered solution (PBS), followed by two more washes with PBS. T-cells previously expanded for 7 days were isolated by immunomagnetic negative enrichment kit (ThermoFisher #11344D) and seeded in triplicate wells at 8.4 × 105cells in 1.2 mL glucose-free RPMI (ThermoFisher #11879020) supplemented with 10 mM [U-13C] glucose (Cambridge Isotope Laboratories #CLM-1396-PK), 10% dialyzed fetal calf serum, 1% SPF, and 1 µg/mL anti-CD28 antibody (BD biosciences #555725). For 72-h treatments, the treatment medium was refreshed 24 h prior to harvest.
Human igg fc fragment
Manufactured by Jackson ImmunoResearch
Sourced in United States
The Human IgG Fc fragment is a laboratory product available from Jackson ImmunoResearch. It is a purified fragment of the Fc region of human immunoglobulin G (IgG). The Fc fragment serves as a tool for various immunological and biochemical applications.
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Lab products found in correlation
Anti cd28 antibody, by BD (1 mentions)
Anti cd3 antibody, by BD (1 mentions)
Mouse ephrinb2 fc, by R&D Systems (1 mentions)
Ephrin b2, by Merck Group (1 mentions)
Rabbit anti human igg fc, by Jackson ImmunoResearch (1 mentions)
Ephrinb2 fc chimera, by R&D Systems (1 mentions)
Ab18181, by Abcam (1 mentions)
Ab3457, by Abcam (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti flag f7425, by Merck Group (1 mentions)
Hrp coupled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Star red, by Abberior (1 mentions)
Anti c myc sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti β tubulin, by Thermo Fisher Scientific (1 mentions)
Anti gfp jl 8, by Takara Bio (1 mentions)
Dyngo 4a, by Abcam (1 mentions)
Star580, by Abberior (1 mentions)
Goat anti mouse igg h l, by Bio-Rad (1 mentions)
Goat anti rabbit igg h l, by Bio-Rad (1 mentions)
2 iminothiolane hydrochloride, by Merck Group (1 mentions)
10 protocols using human igg fc fragment
1
Metabolic Profiling of Activated T Cells
2
Homo- and Heterodimerizing Agents Protocol
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Homo- and heterodimerizing agents AP20187 (Clackson et al., 1998 (link)), AP1887 (Yang et al., 2003 (link)), and AP21967 were obtained from Ariad Pharmaceuticals. Before stimulation, dimerizers were diluted in starving medium used for stimulation to the concentration indicated in the figures. Human IgG Fc fragment (Jackson ImmunoResearch Laboratories, Inc.), mouse ephrinB2-Fc, human ephrinB3-Fc, and human ephrinA5-Fc fusion proteins (R&D Systems) were used for stimulations. For preclustering Fc fragment and ephrin–Fc, fusion proteins were incubated with goat anti–human Fc at a ratio of 5:1 for 30 min at RT.
3
Oligomerization of Ephrin-Fc Fusion Proteins
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Ephrin-A1, ephrin-A5, and ephrin-B2 fused to a human IgG-Fc fragment were purchased from Sigma-Aldrich (Tokyo, Japan; Product #E9902, #E0528, and #E0778, respectively). Before application to the cells, 5 μg of these ephrin-Fc compounds were oligomerized by mixing with 12 μg rabbit anti-human IgG-Fc (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; Code #309-001-008) in 0.1 mL PBS at 4°C for at least 1 h. As a control for these ephrin preparations, a human IgG-Fc fragment (Jackson ImmunoResearch Laboratories; Code #009-000-008) was applied after oligomerization. The final concentration of oligomerized ephrins and IgG-Fc used for each study was 0.5 μg/mL. Human recombinant GH (Sigma-Aldrich; Product #S4776) was used at a final concentration of 0.5 μg/mL.
4
In vivo Thrombopoietin Signaling Activation
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For in vivo stimulation of Thpo signaling, recombinant human Thpo (PEG-rHuMGDF; Kabaya et al., 1996 (link); donated by Kyowa Hakko Kirin Co., Ltd.) was administered. Mice were treated with either 100 µg/kg (i.v.) of PEG-rHuMGDF or human IgG Fc fragment (Jackson ImmunoResearch Laboratories, Inc.). For rescue experiments, Clec2MkΔ/Δ mice were treated for four consecutive days.
5
VEGF, TSP1, and IgG Fc Fragment Protocol
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VEGF (human, recombinant) was from R&D Systems Inc. (Minneapolis, MN). Native human TSP1 was from Athens Research & Technology Inc. (Athens, GA). Human IgG, Fc fragment was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
6
Activation of EphB2 Receptor in Enteric Cultures
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To activate EphB2 receptor in mixed enteric cultures or colonic tissues, we used ephrinB2-Fc chimera (R&D Systems), preclustered with human IgG(Fc) fragment (Jackson ImmunoResearch Laboratories) before the application. To precluster, ephrin-B2-Fc, which connects mouse EphB2 to the Fc portion of the human IgG1 via a polypeptide linker, was mixed with anti-human Fc antibody (1:2) and incubated on ice for 30 min at room temperature. Eleven DIV primary cultures of ENS were stimulated for indicated times with clustered ephrinB2 at a concentration of 0.5 μg/ml. The human IgG(Fc) fragment was subjected to the same treatment and used as control. Cultures were then fixed or not for biochemical analysis or used for electrophysiological recordings.
7
Antibody Validation and Ligand Stimulation
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Antibodies used were as follows: anti-Gulp1 (HPA020587; rabbit polyclonal; Sigma-Aldrich), anti-GFP (JL-8; mouse monoclonal; Clontech), anti-C-Myc (SC-40; mouse monoclonal; Santa Cruz), anti-β-tubulin (480011; mouse monoclonal; Thermo Fisher Scientific), anti-Dyn2 (ab3457; rabbit polyclonal; Abcam), M2 anti-FLAG (F3165; mouse monoclonal; Sigma-Aldrich), anti-FLAG (F7425; rabbit polyclonal; Sigma-Aldrich), and anti-HA (ab18181; mouse monoclonal; Abcam). Secondary antibodies purchased from Jackson ImmunoResearch Laboratories, including HRP-coupled secondary antibodies, were used for Western blots, and fluorescently labeled secondary antibodies were used for immunostaining. For STED imaging, Abberior Star 580 and Abberior Star Red secondary antibodies were purchased from Abberior Instruments.
For soluble ligand stimulation, Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) or mouse ephrinB1-Fc fusion protein (R&D Systems) was incubated with goat anti-human Fc at a ratio of 5:1 for 30 min (Zimmer et al., 2003 (link)). Cells were incubated with the clustered proteins at a final concentration of 2 µg/ml for 30 min at 37°C.
Dynamin inhibitor Dyngo-4a (ab120689; Abcam) was diluted in DMSO.
For soluble ligand stimulation, Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) or mouse ephrinB1-Fc fusion protein (R&D Systems) was incubated with goat anti-human Fc at a ratio of 5:1 for 30 min (Zimmer et al., 2003 (link)). Cells were incubated with the clustered proteins at a final concentration of 2 µg/ml for 30 min at 37°C.
Dynamin inhibitor Dyngo-4a (ab120689; Abcam) was diluted in DMSO.
8
Quantification of IgMRF and IgMRF-α2,6-SIA
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ELISA plates were coated with human IgG Fc fragment (Jackson ImmunoResearch Inc.) at 10 μg/mL for IgMRF detection. 29 , 30 Plasma (1:1000 dilution) was added and followed by an HRP-conjugated goat anti-human IgM antibody (Jackson ImmunoResearch Inc.) for IgMRF measurement (designated as plasma IgMRF). Alternatively, an SNA (Sambucus nigra)-HRP solution (EY Laboratories, Inc.) was coated on the plates for measurement of the α2,6-SIA content of IgM (designated as the IgMRF-α2,6-SIA). 30 Human IgGRF and its α2,6-SIA content were detected with rabbit IgG Fc fragment (Jackson ImmunoResearch Inc.) by a similar method.
9
Characterization of SP141 and FcRn Binding
10
DENV2 E Protein Antibody Analysis
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Human monoclonal antibodies (#753 C6 and #751 A2) against the DENV2 E protein were a kind gift of J. Mongkolsapaya and G. Screaton (Imperial College, UK). Human IgG Fc-fragments were used as control (Jackson immunoResearch, USA). Antibodies for the phenotyping of the macrophages, and corresponding isotypes, were obtained through an unrestricted grant (Immunotools, DE).
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