Mice were infected with L. sigmodontis at 6–8 weeks of age via the tropical rat mite Ornithonyssus bacoti as previously described [13 (link)]. Chronic filarial infection was confirmed by the presence of adult worms within the thoracic cavity at the time of necropsy. Systemic TGFβ 1–3 was depleted on day –3 and day –1 before E. coli challenge by intraperitoneal injection of 100 µg/mouse anti-TGFβ depletion antibody (Clone: 1D11.16.8, BioXCell, West Lebanon, USA), a dose which was previously shown to prevent the protection against diabetes onset by filarial infection in nonobese diabetic mice [3 (link)]. Controls received 100 µg/mouse IgG1 isotype control (Clone: MOPC-21, BioXCell).
Igg1 isotype control
Manufactured by BioXCell
Sourced in United States
The IgG1 isotype control is a laboratory reagent used in flow cytometry and other immunoassays. It serves as a control to establish the appropriate gating and background levels when analyzing specific antibody binding. The IgG1 isotype control provides a non-specific antibody binding profile to differentiate target-specific signals from non-specific background signals.
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5 protocols using igg1 isotype control
1
Depletion of TGFβ exacerbates bacterial infection in filarial-infected mice
2
Phagocytosis of Meningioma Cells by Macrophages
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Meningioma cells were labeled with 1μM CFDA-SE (CFSE) using the Cell Trace CFSE Cell Proliferation Kit (Invitrogen). Macrophages were incubated with PMA for 48 hours. Then macrophages were incubated with 1×106 CFSE-labeled tumor cells in serum-free medium in the presence of 10μg/ml anti-CD47 antibody (B6H12; BioXcell) or 10μg/ml IgG1 isotype control (BioXcell) or PBS for 2hours, respectively. The macrophages were incubated with CD68 antibody to make macrophages red fluorescent protein (RFP) positive before observation. Immunofluorescence assay was performed to observed the phagocytosis. The phagocytic index was calculated as the number of phagocytosed CFSE+ cells per 100 macrophages.
3
Macrophage Depletion in Breast Cancer
4
Bioluminescence Tracking of T. gondii Infection
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The luciferase-expressing Me49-Luc type II T. gondii strain was provided by Laura Knoll (29 (link)). Tissue cysts were obtained from the brains of infected mice. Experimental mice were infected with 10 cysts of T. gondii Me49 orally. Weight was monitored every 48 h for the first 20 days. Parasite burden was analyzed by bioluminescence measurements. Mice were imaged every 48 h for 20 days by i.p. injection of 150 mg of luciferin D (Biosynth AG) per kg of body weight and using a Xenogen IVIS 100 (Caliper Life Sciences). Data was analyzed with the Living Image Software (Caliper Life Sciences) and is expressed in relative light units. Blocking antibodies anti IL-17A (IL-17F) and IgG1 isotype control were purchased from BioxCell. Mice were injected intraperitoneally with 350 μg at day −1 and +5 of T. gondii infection.
5
CD47 Blockade Enhances Macrophage Phagocytosis
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Derived macrophages were harvested and counted by hemocytometer. Once purified, target cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enhance target cell signal strength and then counted. Target cells were cocultured with macrophages at a ratio of 2:1 for 1 h in the indicated treatment condition. CD47 blockade in vitro was achieved using anti-CD47 mAb, for mice MIAP410 (Bio X Cell) was used and for human Hu5F9G4 (Lonza) was used. Isotype control treatment for mouse was IgG1 isotype control (Bio X Cell) and for human was Ultra-LEAF purified IgG4 isotype control (BioLegend). Mouse macrophages were labeled with anti-mouse F4/80 (BioLegend), human macrophages were labeled with anti-human Macrophage Marker (eBioscience). Percent phagocytosis (CFSE+ Macrophages) was quantified by FACS and normalized to PBS control to calculate fold-change in phagocytosis across treatment conditions.
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