21 research outputs found
Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.
Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs
A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies
A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies
Drug Derived Fluorescent Probes for the Specific Visualization of Cannabinoid Type 2 Receptor - A Toolbox Approach
Cannabinoid type 2 receptor (CB2R) is
a fundamental part of the endocannabinoid signaling system (eCB system), and is
known to play an important role in tissue injury, inflammation, cancer and
pain. In stark contrast to its significance, the underlying signaling
mechanisms and tissue expression profiles are poorly understood. Due to its low
expression in healthy tissue and lack of reliable chemical tools, CB2R
visualization in live cells remains uncharted. Here we report the development
of a drug derived toolbox of highly potent, CB2R-selective
fluorescent probes based on reverse design. Extensive validation in several
applications such as CB2R detection in flow cytometry and
time-resolved imaging, and the development of a novel fluorescent-based TR-FRET
assay to generate kinetic and equilibrium binding data demonstrate the high
versatility of our toolbox. These probes are the first to preserve affinity and
efficacy in both human and mouse CB2R, a crucial aspect for
preclinical translatability, and to enable imaging of CB2R internalization in
living cells using confocal microscopy
Highly Specific, Fluorescent Cannabinoid Type 2 Receptor Probes Enable Applications in Microscopy, Flow Cytometry and FRET-based Binding Assays
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite its significance, researchers lack reliable tools to address questions concerning the complex mechanism of CB2R signaling and its downstream consequences, especially in cell-type and tissue-dependent contexts. Herein, we report highly specific CB2R fluorescent probes and their use in a variety of applications: flow cytometry with overexpressing as well as endogenously expressing cells, real-time confocal microscopy of living cells, and a novel FRET-based, CB2R binding assay amenable to high throughput screening.<br /
Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells
Pharmacological modulation of cannabinoid type 2 receptor
(CB2R) holds promise for the treatment of numerous conditions,
including inflammatory diseases, autoimmune disorders, pain, and cancer.
Despite the significance of this receptor, researchers lack reliable tools to
address questions concerning the expression and complex mechanism of CB2R
signaling, especially in cell-type and tissue-dependent context. Herein, we report
for the first time a versatile ligand platform for the modular design of a
collection of highly specific CB2R fluorescent probes, used successfully
across applications, species and cell types. These include flow
cytometry of endogenously expressing cells, real-time
confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based
kinetic and equilibrium binding assays. High CB2R specificity was
demonstrated by competition experiments in living cells expressing CB2R
at native levels. The probes were effectively applied to FACS analysis of
microglial cells derived from a mouse model relevant to Alzheimer’s disease and
to the detection of CB2R in human breast cancer cells
Distributions of calculated BSA monomer signals for the different kits and the different optical systems.
<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p
Correlations of the <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values of the BSA monomer with the difference of the best-fit meniscus from the mean meniscus value, separately for absorbance data sets (A) and interference data sets (B).
<p>The difference of the best-fit meniscus to the mean was calculated separately for each kit, to eliminate offsets due to different sample volumes in each kit, and then merged into groups for the optical systems. Data are shown as a histogram with frequency values indicated in the colorbar. The dotted lines show the theoretically expected dependence of the apparent <i>s</i>-value on errors in the absolute radial position.</p