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Today, in the modern world, allergic diseases, also described as atopic allergies, are classified as a type of multifactorial disorder due to the complex interplay between genetics, environment, and socioeconomic factors that influence the disease's manifestation, severity, and one's predisposition to allergic diseases. It is undeniable that many reported studies have pointed out that the mast cell is one of the leading key players involved in triggering an allergic reaction. To improve our understanding of the molecular and cellular mechanisms underlying allergy, various mast cell lines have been employed in vitro to study the pathogenesis of allergic diseases for the past decades. However, there is no consensus on many fundamental aspects associated with their use, such as the effects of culture media composition and the type of inducer used for cell degranulation. As the standardization of research protocols and disease models is crucial, we present the outcome of a systematic review of scientific articles using three major immortalized in vitro mast cell lines (HMC-1, LAD2, and RBL-2H3) to study allergy. This systematic review described the cell source, culture conditions, inducers used for degranulation, and mediators released for examination. We hope that the present systematic review may help to standardize the use of immortalized in vitro mast cell lines in allergy research and serve as a user's guide to understand the fundamental aspects of allergy as well to develop an effective allergy therapy in the future for the betterment of human good health and wellbeing. [ABSTRACT FROM AUTHOR]