Lamina Propria Mononuclear Cells (LPMCs) were isolated from inflamed colonic surgical specimens from adult subjects with CD who underwent bowel resection at the Mount Sinai Medical Center. LPMCs were isolated according to an established protocol using Dispase II (Roche Diagnostics, Indianapolis, IN) and collagenase (Sigma, St. Louis, MO) treatment.21 (link);22 (link) LPMCs were cultured overnight in serum-free medium. GAC1 (40μg/mL) was then added for 1 hour, and cells were stimulated with TNF-α (10 μg/mL) for 10 min. In-Cell Western Assay (Li-Cor, Lincoln, Nebraska) was performed according to the manufacturer’s instructions.
In cell western assay
Manufactured by LI COR
The In-Cell Western Assay is a quantitative, cell-based assay that allows for the simultaneous detection and quantification of target proteins in their native cellular environment. It combines the specificity of Western blotting with the high-throughput capabilities of microplate-based assays. The assay is performed directly in cultured cells, providing a rapid and efficient method for analyzing cellular signaling pathways, target validation, and drug screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680rd donkey anti rabbit, by LI COR (3 mentions)
Irdye 800cw donkey anti goat, by LI COR (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Collagenase, by Merck Group (2 mentions)
Dispase 2, by Roche (2 mentions)
Phospho iκbα, by Cell Signaling Technology (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Ficoll hypaque, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Celltag 700, by LI COR (1 mentions)
Hdac2, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
On cell western assay, by LI COR (1 mentions)
11 protocols using in cell western assay
1
Isolation and Stimulation of Lamina Propria Mononuclear Cells
2
Isolation and Stimulation of LPMCs
3
Isolation and Activation of PBMCs
4
Quantifying DNA Damage Repair in Cells
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The cells were exposed to 1, 3, 10, 30, and 100 μM RO4929097 and irradiated to 2 and 8 Gy in 96-well tissue culture plates. γ-H2AX assays were done after 1 hour, which was determined as the optimal time point based on preliminary experiments. A quantitative method, In-Cell Western assay (LI-COR Biotechnology) was used. Phosphorylated γ-H2AX foci were taken as a surrogate marker for the function of the DNA damage repair (DDR) pathway. After irradiation and exposure to RO4929097, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were stained for phosphorylated γ-H2AX foci with phosphohistone γ-H2AX (Ser 139) rabbit McAb primary antibody (Cell Signalling Technology, MA) and goat anti-rabbit IRDye secondary antibody (LI-COR Biotechnology). Nuclear and cytoplasmic cell staining was performed with CellTag700 (LI-COR Biotechnology) for the normalization of phosphorylated γ-H2AX foci to the number of cells within each well. The plates were imaged with Odyssey CLx Imager (LI-COR Biotechnology), and the results were analyzed with Image Studio software (LI-COR Biotechnology).
5
Endogenous TRiC/CCT Purification Tag
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To insert a purification tag to the endogenous TRiC/CCT, HEK293T cells were transfected with plasmid encoding eSpCas9(1.1) and sgRNAs targeting CCT5 and ATP1A1 (modified from Addgene no. 86613). These cells were also transfected with linear dsDNAs to act as HDR templates by which to insert 3×FLAG/Spytag at the C terminus of CCT5, as well as the Q118R and N129D ouabain resistance conferring point mutations to ATP1A150 (link),51 (link). Following ouabain selection, the polyclonal cell pool was assessed for FLAG-tagged CCT5 via immunoblotting. Single cells from confirmed pools were seeded into 96-well plates by FACS, and resulting monoclonal lines were again screened via In-Cell Western Assay (LI-COR) followed by western blots. Three positive monoclonal lines were identified and each was further verified by PCR, sequencing, and western blots.
6
Inflammatory Pathway Activation Assay
7
Quantitative In-Cell Western Assay
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Cells were seeded at 5 × 10 4 /well into clear, black-bottom, 96-well plates, incubated overnight at 37°C, and incubated for 1 hour with a 9-point dilution series of each inhibitor, followed by 3.7% formaldehyde fixation, co-staining with pERK and GAPDH antibodies, co-staining with goat secondary antibodies conjugated to IRDye 800CW (for pERK) or IRDye 680RD (for GAPDH), and analysis of staining intensity by In-Cell Western assay (LI-COR Odyssey). Signal intensity for pERK was normalized to GAPDH and the DMSO-treated control samples to generate POC data, which were then plotted versus compound MS# CD-24-0024R1 11 concentration using GraphPad Prism 9 software to generate IC 50 data using a 4-parameter curve fit. See Table S6 for sources of primary antibodies.
8
In-Cell Western Assay for Protein Analysis
9
Quantifying Phagocytosis and Surface Proteins
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In order to detect phagocytosis of bacterial cells or Jurkat T cells by THP-1 macrophages, we analysed these cells by employing the use of specific markers following coculturing of the respective cells. We used a standard LI-COR in-cell Western assay (methanol was used as a permeabilisation agent) [12] to detect bacterial LPS or T cell-associated CD3 in THP-1 macrophages. Rabbit anti-LPS (which recognises lipid A) and anti-CD3 antibodies were used to detect specific targets and a goat anti-rabbit Li-Cor secondary antibody was employed for visualisation purposes.
In order to characterise the presence of galectin-9 and VISTA on the surface of human embryonic cells or Jurkat T cells (galectin-9 only) we used a standard Li-COR on-cell Western assay [12] where the cells were not permeabilised thus measuring only the proteins present on the cell surface.
In order to characterise the presence of galectin-9 and VISTA on the surface of human embryonic cells or Jurkat T cells (galectin-9 only) we used a standard Li-COR on-cell Western assay [12] where the cells were not permeabilised thus measuring only the proteins present on the cell surface.
10
Quantifying Tim-3 and Galectin-9 Expression
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We employed a standard LI-COR in-cell Western (ICW) assay (methanol was used as permiabilization agent) to analyze total Tim-3 and galectin-9 expressions in the studied cells. The in-cell (ICA, also called on-cell) assay was employed to characterize Tim-3 and galectin-9 surface presence in the studied cells. We also used this assay to visualize binding of LAD2 cells to NK cells. IgE-sensitized LAD2 cells were exposed for 5 min to 1 μg/ml, carefully washed with sterile PBS and exposed to LI-COR goat anti-mouse labelled secondary antibody. Following washing with PBS, cells were scanned using a LI-COR Odyssey imaging system (Gonçalves Silva et al., 2016 ).
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