Here we describe the application of mass cytometry to analyze tumor-infiltrating lymphocytes in human melanoma. Mass cytometry is the coupling of flow cytometry and mass spectrometry, which allows for the simultaneous measurement of 40+ cell parameters on a per cell basis. Heavy metal-labeled antibodies can bind to proteins (CD markers, transcription factors, cytokines) on the cell surface and in the cytoplasm/nucleus. As labeled cells pass through the CyTOF, the instrument detects the heavy metals. Combining these signals allows description of melanoma tumor-infiltrating lymphocytes at a greater depth than alternative phenotyping strategies and enables detailed analyses of a variety of cellular parameters, including immune cell lineage, activation status, and functional polarization.We describe here a protocol to measure gene expression, T cell receptor (TCR) sequence, and protein expression by single T cells extracted from melanoma, using 10× Chromium technology. This method includes freezing and thawing of the melanoma infiltrating lymphocytes, staining of cells with fluorescent and barcode-conjugated antibodies, sorting of T cells, and loading the cells on the 10× Chromium Controller. After sequencing, analysis includes quality control, genetic demultiplexing to resolve genetically different samples, and T cell clonality and clustering analysis. Single cell RNA sequencing paints the complete portrait of individual T cells, including their clonality and phenotype, and it reconstructs a complete picture of the T cell infiltrate in a tumor that is represented as cell clustering similar to a pointillism painting.The density of tumour-infiltrating lymphocytes (TILs) in melanoma is correlated with improved clinical prognosis; however, standardized TIL immunotyping and quantification protocols are lacking. Herein, we provide a review of the technologies being utilized for the immunotyping and quantification of melanoma TILs.MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.MicroRNAs (miRs) are small RNA molecules (18-22 nucleotides) that regulate the transcriptome at a post-transcriptional level by affecting the expression of specific genes. This regulatory mechanism is critical to maintain cell homeostasis and specific functions. Aberrant expression of miRs have been associated with pathobiological processes including cancer. click here There are few technologies available that are able to profile whole-genome miR expression using minimal amounts of blood samples and without the need for time-consuming extraction steps. Here, we describe the HTG EdgeSeq miR Whole-Transcriptome Assay (WTA) in serum and plasma samples. To identify specific cell-free miR (cfmiR) patterns we have first focused on the analysis of normal donor samples and have then compared these to patients with cutaneous melanoma. The identification of specific cfmiR for melanoma patients will allow for better patient surveillance during targeted and/or checkpoint inhibitor immunotherapy (CII) treatment.Gut microbiota influence and modulate host immune responses. In preclinical cancer models, mice lacking gut microbiota have a markedly diminished response to immune checkpoint inhibitor therapy. Further, in melanoma patients, specific commensal gut microbiota have been associated with a positive clinical response to immunotherapy. In order to study the gut microbiome and metabolome, we have developed methods for fecal sample collection and processing, microbiome and metabolome profiling, and bioinformatic analysis. This protocol will be a useful tool for interrogating the taxonomic composition and functional output of a melanoma patient's gut microbiome.Multiplex immunoassays simultaneously measure multiple analytes in a single sample providing quantitative data via parallel analyses, which is especially suitable for serum biomarker verification and validation. Multiplex immunoassays demonstrate several advantages over traditional enzyme-linked immunosorbent assays such as increasing productivity, conserving critical reagents and samples, and delivering results quickly. Here we describe the detection of uveal melanoma by magnetic bead-based multiplex immunoassays of serum biomarkers. The biomarker panels evaluated by multiplex immunoassays with high analytical performance demonstrated potential complementary values in detection of uveal melanoma.Canine oral melanoma (COM) is a common oral cancer with aggressiveness and high metastasis. A tumor in a dog's mouth makes it difficult to be observed at the early-clinical stage. Salivary biomarkers may be useful for early detection, prognosis, and monitoring of therapies. In addition, salivary collection is a simple and non-invasive technique. The present study describes how to identify salivary biomarkers in COM using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approaches. Western blot analysis has been used to confirm the protein expression. The sequence of expressed protein from western blot has been verified by LC-MS/MS.Cancer mortality rates are primarily a result of cancer metastasis. Recent advances in microscopy technology allow for the imaging of circulating tumor cells (CTCs) as they extravasate (exit) blood vessels, a key step in the metastasis process. Here, we describe the use of intravital microscopy techniques to image and isolate both extravasating melanoma CTCs and the extravasation-participating endothelial cells. These techniques can be used as a means to study cancer metastasis and as a screening tool for anticancer therapeutics.