The assessment of live/dead cells by means of fluorescence microscopy is a standard technique for assessing cell viability but relies on manual counting, which is laborious and prone to operator bias. Here we present a step-by-step protocol for automated cell viability quantification from two-channel live/dead fluorescence images using a deep learning pipeline built on a U-Net architecture with a ResNet-50 encoder pretrained on ImageNet. The protocol is organized into two tracks: a no-code inference track that applies a pre-trained model to standard live/dead images and an adaptation track for generating custom annotations and retraining the model for new cell types or staining protocols. The pipeline integrates percentile-based normalization, centroid-based (weakly supervised) annotation, patch-based transfer learning, and distance-transform watershed post-processing for instance-level counting. On an independent 25-image test set of human dermal fibroblasts, the model achieved counting R2 values of 0.986 (live) and 0.999 (dead) and a mean absolute viability error of 0.61 percentage points, providing a reproducible framework adaptable to diverse cell types and imaging platforms.
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes the oxidative crosslinking of collagen and elastin in the extracellular matrix, contributing to tissue stiffening and remodeling in the tumor microenvironment. Dysregulated LOX expression has been mechanistically linked to breast cancer invasion, pre-metastatic niche formation, and poor clinical prognosis. Here we describe a complete protocol for evaluating LOX protein expression in formalin-fixed paraffin-embedded (FFPE) human breast tissue sections, comprising (1) hematoxylin and eosin (H&E) staining for morphological assessment and tissue region selection and (2) direct immunofluorescence (IF) detection of LOX using an Alexa Fluor 647-conjugated anti-LOX rabbit recombinant monoclonal antibody (clone EPR4025), which eliminates the need for a secondary antibody step, with antigen retrieval performed under pH 6 citrate buffer conditions. The protocol is validated on ductal carcinoma in situ (DCIS) and DCIS-adjacent breast tissue and is readily adaptable to other FFPE tissue types and directly conjugated fluorescent antibodies.
Stem cell-derived exosomes have gained increasing attention due to their therapeutic potential in various diseases. The successful acquisition of exosomes with the desired therapeutic efficacy requires both the appropriate cultivation of the source stem cells and the use of suitable isolation methods. In this context, serum-free culture approaches have emerged as a key strategy to enhance exosome yield. This chapter explains in detail how to properly grow stem cells, how to adapt them to serum-free culture conditions, and how to get exosomes from these cells.
T cell-mediated therapies are major breakthroughs in the treatment of melanoma. However, clinical hurdles in the application of T cell-mediated therapies in melanoma persist and limit their potential. As such, preclinical research efforts remain focused on their development and optimization. Preclinical research techniques include in vivo evaluation with xenograft mouse models, ex vivo patient tumor explant culture, and in vitro tumor spheroids and melanoma/T cell co-cultures. However, these models lack the appropriate tissue context of melanoma skin cancer. Here, we describe a technique for the incorporation of primary T cells and chimeric antigen receptor (CAR)-T cells with melanoma cells in human skin organoids derived from primary epidermal keratinocytes.
Mechanical properties play a critical role in regulating ovarian function, yet their spatial variation within intact tissue remains poorly understood due to the limitations of conventional mechanical testing techniques. Confocal Brillouin microscopy offers a non-contact and label-free approach for mapping mechanical properties of the intact ovarian tissue without perturbing tissue structure. In fresh ovarian tissue, Brillouin images reveal spatial heterogeneity in mechanical properties across follicles, surrounding stroma, and fluid-filled regions, suggesting underlying differences in cellular organization and extracellular matrix composition. These measurements capture both inter- and intra-follicular variations, providing insight into regional differences in mechanical properties within and between follicles. This chapter outlines the principles of Brillouin microscopy and its application to fresh ovarian tissue, highlighting its capability to resolve microscale mechanical variations in situ. This method provides a foundation for understanding the role of mechanical properties in ovarian function, with potential applications in disease models and reproductive health research.
