Analyzing the Functions of Mast Cells <em>In Vivo</em> Using '<em>Mast Cell Knock-in</em>' Mice

Abstract

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the 'mast cell knock-in' approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years. Video LinkThe video component of this article can be found at

Genetic deletion of mast cell serotonin synthesis prevents the development of obesity and insulin resistance

“…Other reasons for the more modest phenotype in the Tph1 MCKO mice is the time of testing which was after 8 weeks of high-fat diet compared to the 12 weeks for the Kit Tph1−/− mice. The longer duration of high-fat diet feeding in the Kit Tph1−/− mice was utilized to maximize mast cell engraftment as previously suggested 47 . Therefore, sex and duration of the high-fat diet before testing may explain the differences between models.…”

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

“…To track mouse movement and determine displacement, the videos were converted into a sequence of still images using MPEG Streamclip (downloaded from http://www.squared5.com) at 8 frames/s and analyzed using the TrackMate plugin for ImageJ. For analysis of c-Kit expression levels on peritoneal mast cells of C57BL/6J mice, the animals were sacrificed 1 h after injections and a peritoneal lavage with 5 ml of ice-cold PBS was performed to harvest the peritoneal cells as previously described (Gaudenzio et al, 2015). The peritoneal cell suspension was centrifuged, resuspended in 1 ml PBS 1% BSA and 200 μl were subsequently stained with 1:200 anti-c-Kit and anti-FcεRIα antibodies on ice for 15 min, analyzed by flow cytometry on an Accuri C6 flow cytometer (BD Biosciences).…”

“…Histological analysis of peritoneal mast cells was performed based on a published protocol (Gaudenzio et al, 2015). Briefly, 1 ml of peritoneal cell suspension was centrifuged and the pellet resuspended in 100 μl PBS 1% BSA and spun onto microscopy slides using a Cytospin system (Shandon).…”

A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice

“…BMCMCs were obtained through in vitro differentiation of bone marrow precursors in WEHI-3-conditioned DMEM, for at least 5 weeks55. Bone marrow cells derived from 4-week-old Tnfrsf14 −/− , Tnfrsf14 +/+ or Mcpt5-eYFP mice were cultured in WEHI-3-conditioned DMEM (DMEM containing 20% supernatant of WEHI-3 cells, 10% FBS, 50 μM β-mercaptoethanol, 2 mM L -glutamine and 1% antibiotic-antimycotic solution), as a source of IL-3, for 4–5 weeks to generate cell populations that contained >99% bone-marrow-derived cultured MCs (BMCMCs).…”