Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species

BACKGROUND: Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS: Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS: Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study

Designed 2D protein crystals as dynamic molecular gatekeepers for a solid-state device

The sensitivity and responsiveness of living cells to environmental changes are enabled by dynamic protein structures, inspiring efforts to construct artificial supramolecular protein assemblies. However, despite their sophisticated structures, designed protein assemblies have yet to be incorporated into macroscale devices for real-life applications. We report a 2D crystalline protein assembly of C98/E57/E66L-rhamnulose-1-phosphate aldolase (CEERhuA) that selectively blocks or passes molecular species when exposed to a chemical trigger. CEERhuA crystals are engineered via cobalt(II) coordination bonds to undergo a coherent conformational change from a closed state (pore dimensions <1 nm) to an ajar state (pore dimensions ~4 nm) when exposed to an HCN(g) trigger. When layered onto a mesoporous silicon (pSi) photonic crystal optical sensor configured to detect HCN(g), the 2D CEERhuA crystal layer effectively blocks interferents that would otherwise result in a false positive signal. The 2D CEERhuA crystal layer opens in selective response to low-ppm levels of HCN(g), allowing analyte penetration into the pSi sensor layer for detection. These findings illustrate that designed protein assemblies can function as dynamic components of solid-state devices in non-aqueous environments

Controlled Decoration of Single-Walled Carbon Nanotubes with Pd Nanocubes

Although there have been many reports of nanoparticle-decorated single-walled carbon nanotubes (SWCNTs), the morphology of the resulting nanoparticles has lacked consistency and control. The present work demonstrates a process for decorating SWCNTs with Pd nanoparticles that have a tendency toward a selective and distinct cubic shape. SWCNTs were synthesized from an embedded catalyst in a porous anodic alumina (PAA) template. A single galvanostatic electrodeposition created Pd nanowires that contacted the SWCNTs within the pores and Pd nanoparticles that decorated the SWCNTs above the surface of the PAA. A distinct change in potential was observed as nanoparticles nucleated on the SWCNTs. The effects of current density and deposition time on the morphology of the nanoparticles were studied. Optimal deposition parameters yielded Pd nanocubes with smooth and flat facets. The electrochemical response and resulting nanocubic deposits provide insights into the difference in electrochemistry between metallic and semiconducting SWCNTs that are consistent with a disparity in the electron-transfer kinetics. Obtaining Pd nanoparticles of consistent shape that are electrically addressed by SWCNTs provides an improved structure for a variety of nanoparticle applications