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

Abstract

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.

Image analysis reveals cellular fragmentation of Spirulina platensis upon treatment with heavy metal ions

“…Thus, proving that those irregular shapes can also be quantified and compared. Therefore, these methods stand as an example for a cost-effective way of analysis and can potentially eliminate the need for sophisticated instruments such as flow cytometry that are not only expensive but also require frequent calibrations when using cell types of irregular geometry (Hunka et al, 2020). Thus, the findings from this study show that ImageJ can be used to effectively monitor the cells for use in various applications.…”

Betamethasone induces potent immunosuppression and reduces HIV infection in a PBMC in vitro model

“…Data analysis was performed using FlowJo V.10.4.1 software (Tree Star, Ashland, Oregon, USA). Online supplemental figure 1 shows the gating strategy from our previous publication77 and follows other published studies 82 83. In this study, we reported on four activation phenotypes (CD38+HLA-DR+, CD38+HLA-DR−, CD38-HLA-DR+, and CD38−HLA-DR−) and define these as previously described 42 77 80.…”

IgG from Adult Atopic Dermatitis (AD) Patients Induces Nonatopic Neonatal Thymic Gamma–Delta T Cells (γδT) to Acquire IL-22/IL-17 Secretion Profile with Skin-Homing Properties and Epigenetic Implications Mediated by miRNA

“…Although only a small proportion of γδT cells can express IgG receptors in their membranes (CD16/FcγR3A), they are functionally differentiated by CTL activity [ 74 ]. The staining protocol used in our study was based on fluorophores attached to the monovalent affinity-purified Fab fragments directed against the Fc portion of IgG primary antibodies [ 75 ]. Therefore, the staining method hinders CD16 interactions by occupying the Fc portion of the IgG molecule, even allowing for simultaneous CD16 identification in complex cytometry staining panels as described in the literature [ 76 ].…”