Flow cytometry to assess the counts and physiological state of cronobacter sakazakii cells after heat exposure

Producción CientíficaCronobacter sakazakii is an opportunistic pathogen that is associated with outbreaks of neonatal necrotizing enterocolitis, septicaemia, and meningitis. Reconstituted powdered infant formulae is the most common vehicle of infection. The aim of the present study is to gain insight into the physiological states of C. sakazakii cells using flow cytometry to detect the compromised cells, which are viable but non-culturable using plate-based methods, and to evaluate the impact of milk heat treatments on those populations. Dead-cell suspensions as well as heat-treated and non-heat-treated cell suspensions were used. After 60 or 65 °C treatments, the number of compromised cells increased as a result of cells with compromised membranes shifting from the heat-treated suspension. These temperatures were not effective at killing all bacteria but were effective at compromising their membranes. Thus, mild heat treatments are not enough to guarantee the safety of powered infant formulae. Flow cytometry was capable of detecting C. sakazakii’s compromised cells that cannot be detected with classical plate count methods; thus, it could be used as a screening test to decrease the risk derived from the presence of pathogenic viable but non-culturable cells in this food that is intended for newborns’ nutrition.Junta de Castilla y León (projects SAN196/VA07/07, SAN673/VA05/08, and SAN126/09

Activation of human NK cells by Plasmodium-infected red blood cells.

This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-γ. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinized blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown

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

Flow cytometry data standards

<p>Abstract</p> <p>Background</p> <p>Flow cytometry is a widely used analytical technique for examining microscopic particles, such as cells. The Flow Cytometry Standard (FCS) was developed in 1984 for storing flow data and it is supported by all instrument and third party software vendors. However, FCS does not capture the full scope of flow cytometry (FCM)-related data and metadata, and data standards have recently been developed to address this shortcoming.</p> <p>Findings</p> <p>The Data Standards Task Force (DSTF) of the International Society for the Advancement of Cytometry (ISAC) has developed several data standards to complement the raw data encoded in FCS files. Efforts started with the Minimum Information about a Flow Cytometry Experiment, a minimal data reporting standard of details necessary to include when publishing FCM experiments to facilitate third party understanding. MIFlowCyt is now being recommended to authors by publishers as part of manuscript submission, and manuscripts are being checked by reviewers and editors for compliance. Gating-ML was then introduced to capture gating descriptions - an essential part of FCM data analysis describing the selection of cell populations of interest. The Classification Results File Format was developed to accommodate results of the gating process, mostly within the context of automated clustering. Additionally, the Archival Cytometry Standard bundles data with all the other components describing experiments. Here, we introduce these recent standards and provide the very first example of how they can be used to report FCM data including analysis and results in a standardized, computationally exchangeable form.</p> <p>Conclusions</p> <p>Reporting standards and open file formats are essential for scientific collaboration and independent validation. The recently developed FCM data standards are now being incorporated into third party software tools and data repositories, which will ultimately facilitate understanding and data reuse.</p

Comparison of methods for in-house screening of HLA*B57:01 to prevent abacavir hypersensitivity in HIV-1 care

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting