Monocyte adhesion was analyzed as previously described (44 (link)), with some modifications. HLMECs were stimulated with NP or TNF-α for 8 h. The human acute monocytic leukemia cell line THP-1 (Lonza) was prelabeled with Zombie Red fluorescent dye (BioLegend, San Diego, CA) in RPMI 1640 medium for 30 min at 37°C before being added to HLMECs and cocultured for 1 h. Nonadherent cells were removed by gently washing with cold RPMI 1640 medium. The images of adherent THP-1 cells were taken under Cytation 3 cell imaging multimode reader.
Protocol detail
Monocyte Adhesion Assay with HLMECs
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Corresponding Organization : University of Missouri–Kansas City
Other organizations : Nanchang University, University of Kansas Medical Center
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Variable analysis
independent variables
- NP (nanoparticle) treatment
- TNF-α (tumor necrosis factor-alpha) treatment
- Monocyte (THP-1 cell) adhesion to HLMEC (human lung microvascular endothelial cells)
- Incubation time (8 hours for NP or TNF-α stimulation)
- Incubation time (1 hour for THP-1 cell and HLMEC co-culture)
- Cell line (THP-1 human acute monocytic leukemia cell line)
- Cell labeling (Zombie Red fluorescent dye)
- Cell culture media (RPMI 1640)
- Positive control: TNF-α stimulation (to induce monocyte adhesion)
- Negative control: Not explicitly mentioned
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