Protocol to analyze transmigration of human cytotoxic T cells under physiological flow conditions in vitro.

STAR Protoc

Molecular Cell Biology Lab, Department of Molecular Hematology, Sanquin Research, Amsterdam, the Netherlands; Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Leeuwenhoek Centre for Advanced Microscopy (LCAM), Section Molecular Cytology at Swammerdam Instit

Published: September 2022


Category Ranking

98%

Total Visits

921

Avg Visit Duration

2 minutes

Citations

20

Article Abstract

This protocol presents an assay for transmigration analysis of human cytotoxic T cells (CTL) under physiological flow in vitro. We describe detailed analysis steps of human CTL behavior, from adhesion to diapedesis, using live cell imaging which cannot be achieved by in vivo imaging. The flow system is made of 2D plastic surfaces covered by an endothelial monolayer limiting the system but allows for quantitative analysis of CTL behavior with high modifiability. For complete details on the use and execution of this protocol, please refer to Schoppmeyer et al. (2022).

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9254146PMC
http://dx.doi.org/10.1016/j.xpro.2022.101509DOI Listing

Publication Analysis

Top Keywords

human cytotoxic
8
cytotoxic t cells
8
physiological flow
8
ctl behavior
8
protocol analyze
4
analyze transmigration
4
transmigration human
4
t cells physiological
4
flow conditions
4
conditions vitro
4

Similar Publications

(phosphatidylserine synthase 1) encodes an enzyme that facilitates production of phosphatidylserine (PS), which mediates a global immunosuppressive signal. Here, based on in vivo CRISPR screen, we identified PTDSS1 as a target to improve anti-PD-1 therapy. Depletion of in tumor cells increased expression of interferon-γ (IFN-γ)-regulated genes, including , , , and , even in the absence of IFN-γ stimulation in vitro.

View Article and Find Full Text PDF

Interferon-induced senescent CD8 T cells reduce anti-PD1 immunotherapy efficacy in early triple-negative breast cancer.

Sci Transl Med

September 2025

Key Laboratory of Breast Cancer in Shanghai, Department of Breast Surgery, Precision Cancer Medicine Center, Fudan University Shanghai Cancer Center, Shanghai 200032, P. R. China.

Triple-negative breast cancers (TNBCs) lack predictive biomarkers to guide immunotherapy, especially during early-stage disease. To address this issue, we used single-cell RNA sequencing, bulk transcriptomics, and pathology assays on samples from 171 patients with early-stage TNBC receiving chemotherapy with or without immunotherapy. Our investigation identified an enriched subset of interferon (IFN)-induced CD8 T cells in early TNBC samples, which predict immunotherapy nonresponsiveness.

View Article and Find Full Text PDF

The mutagenic translesion synthesis (TLS) pathway, which is critically dependent on REV1's ability to recruit inserter TLS polymerases and the POLζ extender polymerase, enables cancer cells to bypass DNA lesions while introducing mutations that likely contribute to the development of chemotherapy resistance and secondary malignancies. Targeting this pathway represents a promising therapeutic strategy. Here, we demonstrate that the expression of the C-terminal domain (CTD) of human REV1, a ca.

View Article and Find Full Text PDF

Design and synthesis of novel indolinone Aurora B kinase inhibitors based on fragment-based drug discovery (FBDD).

Mol Divers

September 2025

State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, Xinjiang, China.

Aurora kinases are a group of serine/threonine kinases essential for cell mitosis, comprising Aurora A, B, and C. However, the Aurora B is overexpressed in multiple tumors and the aurone has been proved to exhibit potent inhibitory activity against Aurora B kinase by our group. The indolinone was considered as an aurone scaffold hopping analog, and the indolinone-based Aurora B inhibitor library (3577 molecules) was constructed by FBDD strategy.

View Article and Find Full Text PDF

Small Interfering RNA Therapy Targeting the Long Noncoding RNA SMILR for Therapeutic Intervention in Coronary Artery Bypass Graft Failure.

JACC Basic Transl Sci

September 2025

BHF Centre for Cardiovascular Science, Queens Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, the Netherlands. Electronic address: andy.bak

Coronary artery bypass graft (CABG) surgery remains the gold standard of care to prevent myocardial ischemia in patients with advanced atherosclerosis; however, poor long-term graft patency remains a considerable and long-standing problem. Excessive vascular smooth muscle cell (SMC) proliferation in the grafted tissue is recognized as central to late CABG failure. We previously identified SMILR, a human-specific SMC-enriched long noncoding RNA that drives SMC proliferation, suggesting that targeting SMILR expression could be a novel way to prevent neointima formation, and thus CABG failure.

View Article and Find Full Text PDF