Kinetx: a combined flow cytometry assay and analysis software framework to quantitatively measure and categorize platelet activation in real-time

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets’ behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus

Next-generation multiparameter flow cytometry assay improves the assessment of oxidative stress in probiotics

peer-reviewedStability of probiotic products’ potency throughout shelf life is essential to ensure systematic delivery of the dosages required to provide clinically-proven health benefits. Due to the oxygen sensitivity of gut-derived microorganisms, methods for the rapid and accurate monitoring of oxidative stress in probiotics are greatly needed as they can be instrumental to both bioprocess optimization and quality control. This study introduces a next-generation flow cytometry method multiplexing the CellROX® Green and Propidium Iodide probes for the simultaneous measurement of free total reactive oxygen species (ROS) and membrane integrity, respectively. The multiparameter method was compared to the single-parameter assays, measuring either ROS or membrane integrity, for the ability to evaluate the fitness of Lactobacillus rhamnosus GG (LGG) after freeze drying, spray drying and H2O2-mediated oxidative stress. Each stand-alone assay detected only three cell populations, showing either differential membrane integrity (Syto 24+/PI-, Syto 24+/PI+, Syto 24-/PI+) or ROS levels (ROS-, low-ROS, high-ROS), and no correlation could be drawn between these groups. Conversely, the multiparameter method detected up to five physiologically distinct cell populations and allowed the integrated assessment of their membrane integrity and oxidative stress. It also revealed a much larger fitness heterogeneity in LGG as each group of low-ROS and high-ROS cells was found to be formed by a healthier population with an intact membrane (L-ROS/PI-, H-ROS/PI-) and a population with damaged membrane (L-ROS/PI+, H-ROS/PI+). As the CRG probe only detects free unreacted ROS, these populations are suggested to reflect the dynamic lifecycle of ROS formation, accumulation and reactive depletion leading to oxidative damage of macromolecules and consequent cell death. With the stand-alone CRG assay being unable to detect ROS lifecycle, the multiparameter method here presented delivers a superior profiling of the heterogeneity generated by oxidative stress in bacteria and enables a more correct interpretation of CRG fluorescence data. We provide recent examples from literature where the use of a single-parameter fluorescence approach may have led to misinterpret oxidative stress data and eventually draw erroneous conclusions

Combined Use of Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses Is a Powerful Diagnostic Tool of Active Tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents

Combined Use of Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses Is a Powerful Diagnostic Tool of Active Tuberculosis

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infectio

Erythrocyte complement receptor 1 (CR1) expression level is not associated with polymorphisms in the promoter or 3' untranslated regions of the CR1 gene

Complement receptor 1 (CR1) expression level on erythrocytes is genetically determined and is associated with high (H) and low (L) expression alleles identified by a HindIII restriction fragment-length polymorphism (RFLP) in intron 27 of the CR1 gene. The L allele confers protection against severe malaria in Papua New Guinea, probably because erythrocytes with low CR1 expression, are less able to form pathogenic rosettes with Plasmodium falciparum-infected erythrocytes. Despite the biological importance of erythrocyte CR1, the genetic mutation controlling CR1 expression level remains unknown. We investigated the possibility that mutations in the upstream or 3′ untranslated regions of the CR1 gene could control erythrocyte CR1 level. We identified several novel polymorphisms; however, the mutations did not segregate with erythrocyte CR1 expression level or the H and L alleles. Therefore, high and low erythrocyte CR1 levels cannot be explained by polymorphisms in transcriptional control elements in the upstream or 3′ untranslated regions of the CR1 gene

Combined Boyden-Flow Cytometry Assay Improves Quantification and Provides Phenotypification of Leukocyte Chemotaxis

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response

Bisulfite-free analysis of 5MeC-binding proteins and locus-specific methylation density using a microparticle-based flow cytometry assay

DNA methylation analysis is emerging as a new technique with potential capabilities for early cancer detection. However, current state-of-the-art techniques are not easily translatable into routine clinical methods. Herein we describe a bead-based flow cytometry assay which combines DNA hybridization to microparticles with 5MeC-specific proteins/antibodies. These assays can be used to study the binding properties of current and emerging 5MeC-binding proteins and may also have potential in the measurement of 5MeC density in clinical samples for cancer detection

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing