Abstract
Blood endothelial cells (ECs) constitute the primary physical barrier to be crossed by circulating leukocytes, once attracted to a site of ongoing inflammation/infection. Upon a pro-inflammatory stimulus, such as tumor necrosis factor (TNF), ECs upregulate adhesion molecule expression to favor the adhesion and, subsequently, the transendothelial migration of the attracted lymphocytes. To address the ability of a cell to transmigrate through a monolayer of ECs, the classical transmigration assay is usually performed (Muller and Luscinskas, 2008). In the present protocol, adapted from Safuan et al. (2012), we describe an in vitro assay for assessing the functionality of the second step of the transendothelial migration, i.e., the firm adhesion of peripheral blood mononuclear cells (PBMCs) to ECs, under static conditions. By pre-incubating primary human umbilical cord ECs (HUVECs) with either innate lymphoid cell progenitors (ILCPs) or TNF, we were able to upregulate adhesion molecules on the EC surface. Then, by adding total PBMCs, we were able to both quantitatively and qualitatively analyze the cellular subtype and number of PBMCs that adhered to the pre-treated ECs. The important advantage of this technique is the possibility to perform functional studies on ECs biology since, differently from transwell-based strategies, it allows a good recovery of ECs at the end of the assay. Overall, this assay enables to interrogate how/if different stimulations/cell types can influence EC ability to retain PBMCs in vitro, under static conditions. Graphic abstract: The workflow of the Static Adhesion Assay.
Highlights
1. 24-well flat bottom polystyrene plate (Corning, Costar, catalog number: CLS3524) 2. 48-well flat bottom polystyrene plate (Corning, Costar, catalog number: CLS3548) 3. 1.5 mL Eppendorf tubes 4. 50 mL Falcon tube 5. 0.22 μm filter 6. 75 cm[2] Tissue Culture Flasks (TPP, catalog number: TPP 90075) 7. 5 mL round bottom tubes with snap cap (Corning, Costar, catalog number: 352058) 8
To address the ability of a cell to transmigrate through a monolayer of endothelial cells (ECs) in vitro, the transwell-based transmigration assays are the most commonly performed (Muller and Luscinskas, 2008)
BD FACSAria II (BD Biosciences) – or any other fluorescence-activated cell sorter equipped with a blue laser 4
Summary
Thaw cryopreserved ECs at 37°C in a water bath, and transfer the cells to a Falcon tube containing 10 mL of complete EGM. 4. Add 10 mL of complete EGM, collect the cell suspension, and transfer it to a Falcon tube. 5. Discard the supernatant, resuspend the cell pellet in 1-2 mL of complete EGM. Once the cells are adherent, remove the medium, and wash ECs with 1 mL of PBS 1×. B. Positive control: 500 μL of complete EGM containing 20 ng/mL of TNF. C. Experimental setting: 500 μL of complete EGM containing ILCPs, at 1:1 ratio. 5. Detach ECs by adding 150 μL of Accutase and incubate at 37°C during 5 min. Transfer the cell suspension to a 5 mL round bottom tube, and wash with 3 mL of PBS 1×. Centrifuge the cells at 200 × g for 5 min at RT
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