Recombinant TcdB (from C. difficile strain VPI 10463) and TcdA were expressed in Bacillus megaterium as previously described49 (link) and purified as His6-tagged proteins. TcdB1-1830 was cloned into pHis1522 vector (MoBiTec) and expressed in B. megaterium. TcdB1831-2366, TcdB1501-2366, and TcdB1114-1835 were cloned into pGEX-6P-1 or pET28a vectors and purified as GST-tagged or His6-tagged proteins in E. coli. NG2-EC (P1 and P2) was expressed in HEK293 cells, purified from medium with DEAE-Sepharose columns, and eluted with a gradient buffer (NaCl from 0.2 to 0.8 M, 50 mM Tris-Cl, pH 8.6) as previously described50 (link). Recombinant human proteins were purchased from ACRO Biosystems (IgG1 Fc and FZD2-CRD-Fc), R&D Systems (FZD1-CRD-Fc, FZD5-CRD-Fc, and FZD7-CRD-Fc), and Sino Biologics (PVRL3-EC).
Igg1 fc
Manufactured by R&D Systems
Sourced in United States, Japan
IgG1-Fc is a recombinant protein consisting of the Fc region of human IgG1. It can be used as a control or reference standard in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Fzd2 crd fc, by R&D Systems (2 mentions)
Phis1522 vector, by MoBiTec (2 mentions)
Atr 107, by Pfizer (2 mentions)
Cd3 bv510, by BioLegend (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd19 pe cy7, by BioLegend (1 mentions)
Recombinant human il 21, by Thermo Fisher Scientific (1 mentions)
Cytofix buffer, by BD (1 mentions)
Pstat3 pe, by BD (1 mentions)
Cd19 bv510, by BioLegend (1 mentions)
Igd apc cy7, by BioLegend (1 mentions)
Cd43 microbead, by Miltenyi Biotec (1 mentions)
Goat anti human igm, by Jackson ImmunoResearch (1 mentions)
Cd38 bv421, by BD (1 mentions)
Cd27 pe cy7, by Thermo Fisher Scientific (1 mentions)
7 aad percp, by BD (1 mentions)
Affi gel blue beads, by Bio-Rad (1 mentions)
Vitronectin, by Merck Group (1 mentions)
High binding microtiter plates, by Corning (1 mentions)
11 protocols using igg1 fc
1
Purification of Recombinant C. difficile Toxins
2
Purification of Recombinant C. difficile Toxins
3
STAT3 Phosphorylation in T and B Cells
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Phosphorylation of STAT3 by CD4+ T cells and CD19+ B cells was determined by phospho-specific flow cytometry. In brief, PBMCs were stained with CD3 BV510 (Biolegend) and CD19 Pe-Cy7 (Biolegend) for 30 min at RT in the dark. Next, the cells were incubated for 30 min at 37°C with various concentrations of the humanized anti-IL-21R antibody ATR-107 (Pfizer) or isotype-matched control (IgG1-Fc, R&D systems) followed by stimulation with recombinant human IL-21 (100 ng/ml, eBioscience) or recombinant human IL-6 (100 ng/ml, PeproTech, Rocky Hill, NJ, USA) for 15 min at 37°C. Cells were fixed for 10 min with Cytofix buffer (BD Biosciences) at 37°C and permeabilized 30 min in 1 ml methanol 90% at −20°C. Next, samples were stained with CD4 BV421 (Biolegend) and pSTAT3 PE (BD Biosciences). STAT3 phosphorylation was calculated as the median fluorescence intensity.
4
BMP-2 and EPHA6-Fc Signaling Protocol
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Recombinant human BMP‐2 was a gift from Bioventus LLC. BMP‐2 was given at 100 ng/mL in the experiments unless otherwise indicated. Recombinant human EPHA6‐Fc and IgG1‐Fc were procured from R&D Systems (Bio‐Techne).
5
Plasmablast Formation from B Cells
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B cell stimulation with a minor cocktail of stimuli was performed to study the effect of IL-21, co-stimulation, and BCR activation on plasmablast formation. CD19+ B cells were isolated via CD43 negative selection with CD43 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (purities ≥85%). B cells were incubated with anti-IL-21R antibody ATR-107 (10 μg/ml, Pfizer) or isotype-matched control (10 μg/ml IgG1-Fc, R&D systems). Next, cells were stimulated with 5 μg/ml soluble anti-CD40 (Bioceros, Utrecht, The Netherlands), 10 μg/ml goat-anti-human IgM (Jackson Immunoresearch, West Grove, PA, USA) and human recombinant IL-21 (100 ng/ml, eBioscience). Subsequently, the presence of plasmablasts on day 0 and the differentiation of memory B cells into plasmablasts on day 8 were determined with flow cytometry. Plasmablasts were defined as CD19posCD27highCD38high cells (16 (link)). The following MoAbs were used: CD19 BV510 (Biolegend), CD27 Pe-Cy7 (eBioscience), IgD APC-Cy7 (Biolegend), and CD38 BV421 (BD Biosciences). In addition, viability staining with 7-AAD PerCP was performed (BD Biosciences).
6
Jag1-Fc Fusion Protein Injection in Treg-Depleted Mice
7
Binding of CD44 to HA and PRG4 Proteins
8
Adhesion Assay for Th1 Cells
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A high binding 96-well plate (Corning) was coated with ICAM-1 (R&D Systems) or IgG1 FC (R&D Systems) (10 μg/ml) for 2 h at 37°C. Non-specific binding was blocked with adhesion buffer (HBSS Ca2+ Mg2+ supplemented with 1% BSA) for 1 h at 37°C. Th1 rep cells were washed twice with PBS, resuspended in pre-warmed, equilibrated adhesion buffer (2 × 106 cells/ml) and starved for 1 h at 37°C and 5% CO2. PMA (10 ng/ml), Ionomycin (1 μg/ml) and CXCL10 (100 ng/ml, Immunotools) were added 10 min before the cell suspension was transferred into the coated wells (50 μl/well). Forty-five minutes after incubation and adhesion at 37°C and 5% CO2, the plate was washed 4 times with 250 μl warm adhesion buffer using an ELX washer according to the manufacturers recommendations. Adherent cells were detached with ice cold PBS/BSA/EDTA and counted using a MACSQuant (Miltenyi Biotec).
9
Phage-ELISA and Indirect ELISA for HER2 Detection
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For phage-ELISA, the wells of 96-well black plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1000 ng of HER2-Fc, streptavidin (Wako, Osaka, Japan), or IgG1-Fc (R&D systems) and blocked with 2% Perfect-Block (MoBiTec, Göttingen, Germany) in PBS for 1 h. Phages (1 × 1010 pfu) were added to each well and incubated for 1 h. After washing the plate, the bound phages were detected with the anti-T7 fiber tail antibody (Merck Millipore) as a primary antibody and the anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA).
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (Ex/Em = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.
10
NK Cell Degranulation Assay with PD-1/PD-L1
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