RESUMEN
The human lung is a complex organ comprised of diverse populations of epithelial, mesenchymal, vascular and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short), which provides a high-yield single cell suspension suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared to four other dissociation protocols. Resulting single cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and non-hematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue with no reduction in viability when compared to previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared to four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.
RESUMEN
Conventional cellular protein detection techniques such as immunocytochemistry and flow cytometry require abundant cells, posing multiple challenges, including difficulty and cost for obtaining enough cells and the potential for clogging the instrument when using flow cytometry. Also, it is challenging to conduct cellular protein imaging and quantification simultaneously from a single experiment. We present a novel 3D platform, which integrates highly biocompatible cell-entrapped alginate hydrogel droplet array with gold-nanoparticle (AuNP)-based metal enhanced fluorescence (MEF), to achieve simultaneous imaging and quantification of proteins in intact cells in a sensitive manner. Compared to 2D immunocytochemistry, this 3D system allows for a higher cell loading capacity per unit area; together with the MEF-based signal enhancement from the embedded AuNPs, sensitive protein quantification was realized. Furthermore, compared to flow cytometry, this platform allows for protein imaging from individual cells. Taking the detection of EpCAM protein in ovarian cancer cells as a model, we optimized the AuNP size and concentration for optimal fluorescent signals. The 5 nm AuNPs at 6.54 × 1013 particles/mL proved to be the most effective in signal enhancement, providing 2.4-fold higher signals compared to that without AuNPs and 6.4-fold higher signals than that of 2D immunocytochemistry. The number of cells required in our technology is 1-3 orders of magnitude smaller than that of conventional methods. This AuNP-embedded hydrogel platform combines the benefits of immunocytochemistry and flow cytometry, providing increased assay sensitivity while also allowing for qualitative analysis through imaging, suitable for protein determination in a variety of cells.
Asunto(s)
Hidrogeles , Nanopartículas del Metal , Oro , FluorescenciaRESUMEN
BACKGROUND: Atrial fibrillation (AF) is associated with extracellular matrix (ECM) remodelling and often coexists with myocardial fibrosis (MF); however, the causality of these conditions is not well established. OBJECTIVE: We aim to corroborate AF to MF causality by quantifying left atrial (LA) fibrosis in cardiac magnetic resonance (CMR) images after persistent rapid ventricular pacing and subsequent AF using a canine model and histopathological validation. METHODS: Twelve canines (9 experimental, 3 control) underwent baseline 3D LGE-CMR imaging at 3T followed by insertion of a pacing device and 5 weeks of rapid ventricular pacing to induce AF (experimental) or no pacing (control). Following the 5 weeks, pacing devices were removed to permit CMR imaging followed by excision of the hearts and histopathological imaging. LA myocardial segmentation was performed manually at baseline and post-pacing to permit volumetric %MF quantification using the image intensity ratio (IIR) technique, wherein fibrosis was defined as pixels > mean LA myocardium intensity + 2SD. RESULTS: Volumetric %MF increased by an average of 2.11 ± 0.88% post-pacing in 7 of 9 experimental dogs. While there was a significant difference between paired %MF measurements from baseline to post-pacing in experimental dogs (P = 0.019), there was no significant change in control dogs (P = 0.019 and P = 0.5, Wilcoxon signed rank tests). The median %MF for paced animals was significantly greater than that of non-paced dogs at the 5-week post-insertion time point (P = 0.009, Mann Whitney U test). Histopathological imaging yielded an average %MF of 19.42 ± 4.80% (mean ± SD) for paced dogs compared to 1.85% in one control dog. CONCLUSION: Persistent rapid ventricular pacing and subsequent AF leads to an increase in LA fibrosis volumes measured by the IIR technique; however, quantification is limited by inherent image acquisition parameters and observer variability.