A live cell biosensor protocol for high-resolution screening of therapy-resistant cancer cells

by Viral D. Oza, Colin S. Williams, Jessica S. Blackburn

The Genetically Encoded Death Indicator (GEDI) is a ratiometric, dual-fluorescence biosensor that enables real-time detection of cell death through calcium influx. Originally developed for use in neurodegeneration models, GEDI can be applied to cancer cells to quantify therapy-induced death at single-cell resolution. This protocol details how to generate GEDI-expressing cancer cell lines, empirically determine stress-induced GEDI thresholds using radiation or chemotherapeutic agents, and perform time-resolved imaging and image analysis to track cell fate. This workflow is optimized for high-throughput drug and radiation screening in heterogeneous populations and is especially useful for identifying chemo- and radio-resistant subclones. Key limitations include the need for empirical GEDI threshold calibration for each treatment condition and careful standardization of imaging parameters. The protocol outputs include GEDI ratio values, single-cell time-of-death annotations, and whole-cell morphological data in parallel, which can be linked to downstream applications such as FACS-based isolation of live or dying subpopulations, transcriptomic profiling of resistant clones, or in vivo validation using xenografts or organotypic slice culture.

Parenterally administered pegbovigrastim alters leukocyte counts, granulocyte functions, and uterine cell population in healthy postpartum dairy cows

by Dinesh Dadarwal, Kira Crooks, Patricia Lainetti, Ryan Dickinson, Khawaja Ashfaque Ahmed, Colin Palmer

This study aimed to evaluate the effects of a single postpartum administration of pegbovigrastim, a recombinant bovine granulocyte colony-stimulating factor (rG-CSF), on peripheral leukocyte profiles, granulocyte function, and uterine cytology in healthy Holstein dairy cows. We hypothesized that rG-CSF would enhance leukocyte counts and granulocyte function without adversely affecting uterine immune cell composition. Twenty-three cows between 19–23 days in milk were randomly assigned to receive either rG-CSF (n = 12) or saline (n = 11). Blood samples were collected on the day of injection and on Days 3, 6, 10, and 21 post-treatment to assess total and differential leukocyte counts. Granulocyte phagocytosis of fluorescein isothiocyanate (FITC)-labeled Staphylococcus aureus and oxidative burst capacity following PMA stimulation were evaluated using flow cytometry. Vaginoscopy and transrectal ultrasound examinations were conducted at each time point, and uterine cytobrush samples were collected from a subset of cows for cytological analysis. Compared to controls, rG-CSF-treated cows exhibited a significant (2–3 fold) increase in total leukocytes and neutrophils (P P P = 0.04) and phagocytic activity as well as capacity (P = 0.01) that peaked on Days 3 and 6 post-treatment, respectively, following rG-CSF treatment. Furthermore, uterine samples from treated cows showed higher proportions of neutrophils (Days 6, 10, and 21) and macrophages (Day 10) compared to controls (P