Endocytic trafficking and recycling are fundamental cellular processes that control essential functions such as signaling protein complexes transport and membrane identity. The small GTPase Rabs are indispensable component of the endosomal recycling machinery. The Rabs bind to effectors to mediate their functions, such as protein sorting and degradation, membrane tethering or lipid modification, and organelle motility. Due to the complex and dynamic nature of endosomal compartments and tracking route, detailed multiparametric analyses of three-dimensional data by quantitative methods are challenging. Here, we describe a detailed time-lapse imaging protocol designed for the quantitative tracking of single endosomal vesicles, using GFP-Rab4-positive recycling endosomes. This method permits automated tracking of single endocytic vesicles in three-dimensional live cell imaging, allowing the study of multiple parameters such as abundance, speed, directionality, and subcellular localization, as well as protein colocalization. This protocol can be broadly used in any kind of cellular models, under various contexts, including growth factors stimulation, gene knockdowns, drug treatments, and is suitable for high throughput screens.

Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na⁺/K⁺ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera’s focal plane and the microscope’s field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching–thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.

Xenograft models, and in particular the mouse xenograft model, where human cancer cells are transplanted into immunocompromised mice, have been used extensively in cancer studies. Although these models have contributed enormously to our understanding of cancer biology, the zebrafish xenograft model offers several advantages over the mouse model. Zebrafish embryos can be easily cultured in large quantities, are small and easy to handle, making it possible to use a high number of embryos for each experimental condition. Young embryos lack an efficient immune system. Therefore the injected cancer cells are not rejected, and the formation of primary tumors and micrometastases is rapid. Transparency of the embryos enables imaging of primary tumors and metastases in an intact and living embryo. Here we describe a method where GFP expressing tumor cells are injected into pericardial space of zebrafish embryos. At four days post-injection, the embryos are imaged and the formation of primary tumor and distant micrometastases are analyzed.

CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD⁺) metabolism. Through its major activity, the hydrolysis of NAD⁺, CD38 helps maintain the appropriate levels of this molecule for all NAD⁺-dependent metabolic processes to occur. Due to current advances and studies relating NAD⁺ decline and the development of multiple age-related conditions and diseases, CD38 gained importance in both basic science and clinical settings. The discovery and development of strategies to modulate its function and, possibly, treat diseases and improve health span put CD38 under the spotlights. Therefore, a consistent and reliable method to measure its activity and explore its use in medicine is required. We describe here the methods how our group measures both the hydrolase and cyclase activity of CD38, utilizing a fluorescence-based enzymatic assay performed in a plate reader using 1,N⁶-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) as substrates, respectively.

Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [³⁵S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.

The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [³H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are incubated with radiolabelled [³H-methyl]-choline in medium for 15 min (pulse). The [³H-methyl]-choline is then rapidly removed and incubation continued in the presence of non-radioactive medium (chase). Cellular distribution of [³H-methyl] is then determined by cell fractionation and measurement of radioactivity in the lipid and non-lipid cellular components.

Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of ⁵⁹Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.

Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis (Greenberg et al., 2011). For example, neutral lipid storage is enhanced upon increased synthesis or uptake of lipid species. On the other hand, extracellular signals can enhance the release of lipid species packaged within neutral LDs. Thus, the investigation of topics involving lipid metabolism may require the assessment of cellular neutral lipid content. In this protocol, we describe the use of the fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) to facilitate quantification of neutral lipid content by flow cytometry and observation of LDs by microscopy.

Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including ¹³C₆-glucose and ¹³C₅,¹⁵N₂-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors.