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Cell engineering Cell imaging Cell isolation and culture Cell metabolism
Cell movement Cell signaling Cell staining Cell structure Cell Transplantation Cell viability Cell-based analysis Model organism culture Organelle isolation Single cell analysis Tissue analysisAn Unbiased CRISPR-Cas9 Screening Method for the Identification of Positive and Negative Regulatory Proteins of Cell Adhesion
Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor–controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion–regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.
Graphical abstract:
Static Adhesion Assay for Human Peripheral Blood Mononuclear Cells
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.
Analysis of B Cell Migration by Intravital Microscopy
During immune responses, B cells home to lymph nodes (LN), where they encounter antigens. Homing starts with capture and L-selectin-dependent rolling on the activated endothelium of high endothelial venules (HEV). After recognition of chemokines presented on HEV, activation of B cell integrins occurs mediating firm arrest. Subsequently, B cells crawl to the spot of extravasation to enter the LN. Extravasation can be visualized and quantified in vivo by intravital microscopy (IVM) of the inguinal LN. Here, we describe an established protocol that permits detailed in vivo analysis of B cell recruitment to LN under sterile inflammatory conditions. We describe data acquisition, exportation, quantification, and statistical analysis using specialized software. IVM of LN is a powerful technique that can provide a better understanding of B cell migratory behavior during inflammation in vivo.
Preparation of Actinoplanes missouriensis Zoospores and Assay for Their Adherence to Solid Surfaces
Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells
Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay
