Elliott, J. T. | Search bioRxiv

bioRxiv2025

Preservation of soluble enhanced green fluorescent protein (EGFP) within fixed NIH 3T3 fibroblasts

Quantitative imaging of cytoplasmic green fluorescence protein (GFP) in fixed cells can be unreliable if the fixing process does not preserve the total fluorescence intensity level or the spatial location relative to the living cells. In this study, we examine the effect of fixatives (formaldehyde, disuccinimidyl glutarate (DSG), dithiobis(succinimidyl) propionate (DSP) and m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS)), fixative buffers and the cross-linking times on the fluorescent intensity of soluble enhanced GFP (EGFP) expressed within NIH 3T3 cells. The total fluorescence intensity within individual cells during the fixation process was measured in an automated fluorescence microscope. Our results show that choice of fixative, the fixing solution and the cross-linking time were important for minimizing EGFP losses during fixing. The optimal fixation condition for these cells was identified to be the MBS cross-linker in a microtubule stabilizing buffer. After an 8 h fixation, greater than 90 % of the initial GFP fluorescence within cells was preserved. This was 3-fold higher than the GFP fluorescence remaining when the cells were fixed with 1 % paraformaldehyde in PBS. MBS treated cells could be permeabilized with 0.05 % Triton X-100 with little additional loss in fluorescence intensity. The MBS fixative could also preserve soluble fluorescent proteins in suspension cells, suggesting the fixation results are not cell line-specific. Direct imaging of the fixation process on fluorescent reporter cells provided insight into the effect of chemical fixatives on biological cells and can facilitate identification of fixation protocols that are fit-for-purpose for quantitative measurements.

cell biology↗

bioRxiv2023

Pharmacokinetic profile of the synthetic mu-opioid receptor agonist Dermorphin-IRDye(R)800CW and its feasibility as a biomarker for opioid use disorder

BackgroundOpioid use disorder (OUD) affects more than 14 million Americans and poses a high risk of relapse, overdose, and death. Current treatments are not tailored to individual needs and do not monitor the effectiveness of the medication. We propose a novel method to measure the occupancy of mu opioid receptors (MOR), which are key targets for opioid pharmacotherapy, in peripheral tissues with high MOR density. We developed a fluorescent peptide agonist that binds to MOR and can be detected by non-invasive point-of-care techniques. We present in vitro and in vivo results that demonstrate the feasibility and potential of this method to assess MOR availability and treatment efficacy in OUD patients. MethodsA new fluorescent-labeled synthetic peptide agonist [Lys7]Dermorphin-IRDye800CW, called DRM-800, was synthesized and characterized in vitro to evaluate binding and internalization. Wildtype and MOR knock-out mice were used to quantify plasma kinetics and, using a cyromacrotome, fluorescence images were acquired post-mortem on whole-body sections 150 um apart. These volumes were used to compare in vivo enhancement of MOR-rich structures. ResultsIn vitro assays and microscope visualization of DRM-800 showed high MOR-affinity and rapid, robust internalization. Plasma half-life following intravenous injection in mice was 8-12 minutes. Specific binding by tissue structures of interest, measured by the ratio of relative fluorescent units in wild-type vs. MOR knockout mice showed high binding in dorsal root ganglia, spiral ganglia and trigeminal ganglion, as well as in the small and large intestine. ConclusionsThe pharmacokinetics and distribution, binding kinetics and rapid internalization suggests that MOR-specific fluorescence enhancement corresponding to opioid rich structures could serve as a potential biomarker in opioid use disorder.

bioengineering↗