Immunophenotyping of Humanized Mice

Mononuclear cells from the peripheral blood and spleens of humanized mice and PBMCs from patients were reacted with fluorescent antibodies (BD Biosciences, San Diego, CA, USA) against CD4 (clone RPA-T4), CD8 (clone SK1), CD19 (clone HIB19), CD25 (clone M-A251), CD14 (clone MφP9), IL-17 (clone SCPL1362), phosphorylated STAT3 (Tyr705, pSTAT3, clone 4/P-STAT3), and collagen type I (COL1A1, R&D Systems, Minneapolis, Minn). Prior to intracellular staining, the cells were restimulated for 4 h with phorbol myristate acetate (25 ng/mL, Sigma-Aldrich) and ionomycin (250 ng/mL, Sigma-Aldrich) in the presence of GolgiSTOP (BD Biosciences, San Diego, CA, USA). Intracellular staining was performed using a kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Flow cytometry was performed using a FACSCalibur instrument (BD Biosciences, San Diego, CA, USA). Mouse antibodies targeting human endothelin receptor A (ETAR) were measured using a commercial enzyme-linked immunosorbent assay kit (MyBioSource Inc., San Diego, CA, USA). Detailed measuring protocols followed the ref. ^{19 (link)}.

Free full text: Click here

Park M.J., Park Y., Choi J.W., Baek J.A., Jeong H.Y., Na H.S., Moon Y.M., Cho M.L, & Park S.H. (2022). Establishment of a humanized animal model of systemic sclerosis in which T helper-17 cells from patients with systemic sclerosis infiltrate and cause fibrosis in the lungs and skin. Experimental & Molecular Medicine, 54(9), 1577-1585.

Publication 2022

COL1A1 phorbol myristate acetate ionomycin GolgiSTOP FACSCalibur instrument enzyme-linked immunosorbent assay kit

Antibodies Cd25 Cells Clone Collagen type i Endothelin receptor Enzyme linked immunosorbent assay Flow cytometry Fluorescent antibodies Human Il 17 Intracellular Ionomycin Mouse Patients Peripheral blood mononuclear cells Phorbol myristate acetate Stat3

Corresponding Organization :

Other organizations : Catholic University of Korea, Seoul St. Mary's Hospital

Top 5 similar protocols

Variable analysis

independent variables

Humanized mice mononuclear cells from peripheral blood and spleens
PBMCs from patients

dependent variables

CD4 expression
CD8 expression
CD19 expression
CD25 expression
CD14 expression
IL-17 expression
Phosphorylated STAT3 (Tyr705, pSTAT3) expression
Collagen type I (COL1A1) expression
Mouse antibodies targeting human endothelin receptor A (ETAR)

control variables

Phorbol myristate acetate (25 ng/mL)
Ionomycin (250 ng/mL)
GolgiSTOP

controls

Positive control: Cells were restimulated with phorbol myristate acetate and ionomycin prior to intracellular staining.
Negative control: Not explicitly mentioned.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!