Viable cells were recognized based on absence of DAPI or Aqua dye. single-cell analysis Use of a membrane barrier and cytoskeletal inhibitors to block trogocytosis Fluorescence microscopy and circulation cytometry to visualize transfer of membrane Calcium-Sensing Receptor Antagonists I fragments Publishers note: Starting any experimental protocol requires adherence to local institutional recommendations for laboratory security and ethics. Trogocytosis is an active transport mechanism by which one cell components a plasma membrane fragment with inlayed molecules from an adjacent cell inside a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting important experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. == Before you begin == This protocol details methods from several orthogonal methods to demonstrate that human being NK cells acquire CD9 from ovarian tumor cells by trogocytosis (Gonzalez et al., 2021). While we make use of a human being system, the methods are equally relevant to studies in additional varieties. Each method requires creating cocultures between NK and tubo-ovarian high grade serous carcinoma (HGSC) cell lines. Most frequently we use the OVCAR4, and NK-92 cell lines. However, we have performed these methods with additional HGSC ovarian tumor cell lines (Kuramochi and TYK-nu), as well as with cell lines derived from additional malignancies (colon (HCT116 and CaCo), epidermoid/pores and skin (A431) and breast (MCF7)). In addition, we have used the NKL cell collection and main NK cells from peripheral blood mononuclear cells (PBMCs). Systems used include mass cytometry (Cytometry by Time-Of-Flight (CyTOF)), fluorescence-based circulation cytometry and fluorescence microscopy. For each technology steps to prepare and analyze samples are provided. The methods have been developed from previous work by us while others and enable a researcher to demonstrate the transfer by trogocytosis of a protein of interest between two different cell types in both human being and murine systems (Daubeuf et al., 2010;Gary et al., 2012;Gonzalez et al., 2021;Hamieh et al., 2019;Li et al., 2019;Reed and Wetzel, 2019;Schriek et al., 2022;Tilburgs et al., 2015;Uzana et al., 2012;Vanherberghen et al., 2004;Hasim et al., 2022). Notice:The system chosen for this protocol offers specific relevance to the field of immuno-oncology. However, it is widely relevant to many cell types and thus facilitates the investigation of trogocytosis in additional systems. == Institutional permissions == Peripheral blood samples from anonymous healthy donors were from the Stanford Blood Center (Palo Alto, CA, USA). == Culturing NK cell lines == Timing: 1.52 weeks The protocol below describes specific methods for culturing human being NK cell lines (NK-92 and NKL) from ABCC4 frozen stocks. It includes press preparation, cell thawing, counting, and viability dedication (Gonzalez et al., 2021,2022). Both cell lines use the same tradition conditions. CRITICAL:Successful growth of NK cell lines is definitely highly dependent on the quality of IL-2 used in the tradition media. The activity of IL-2 can change between lots. The activity of the IL-2 we use as calibrated by the manufacturer has an ED50of 0.050.25 ng/mL inside a murine T cell proliferation assay. Its specific activity is definitely 9.1 106 Calcium-Sensing Receptor Antagonists I IU/mg when calibrated against recombinant IL-2 WHO International Standard. Please consult vendors’ specifications using the guidelines described as helpful tips. We have found R&D Systems to be a reliable source of recombinant human being IL-2 protein. Notice:IL-2 is critical for both NK cell viability and proliferation and Calcium-Sensing Receptor Antagonists I is added new to media utilized for NK cell ethnicities (Kotzur et al., 2022). Notice:For supplemented RPMI-1640 + IL-2: freshly thawed IL-2 stock solution is definitely added immediately before each use and all subsequent media changes for NK cells in tradition should likewise become with supplemented RPMI-1640 comprising freshly thawed IL-2. Notice:Supplemented RPMI (without IL-2) can be stored at 4C for up to 3 months. Notice:Good Practice per Environmental Health and Security at Stanford, the lab space temperature should be managed at 20C26C. Notice:Cells are thawed and diluted quickly into supplemented press + IL-2 to minimize harmful effects of DMSO. Note:Keep time that NK cells are in unsupplemented RPMI-1640 to an absolute minimum. Notice:For accurately distinguishing live from deceased cells, it is essential to use unsupplemented press as Calcium-Sensing Receptor Antagonists I trypan blue binds strongly.