Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining)

Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A

<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h^-1with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O₂in the inlet gas were quantified by ¹³C-MFA. Metabolic flux ratios from fractional [U-¹³C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of <it>S. cerevisiae</it>.</p> <p>While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO₂. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.</p> <p>Conclusion</p> <p>¹³C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.</p

Toll-like receptor modulation of disease relevant B cell responses

B cells are becoming increasingly recognized as important immune regulatory cells. Depending on their context of activation, the balance of B cell production of proinflammatory and anti-inflammatory cytokines is affected. Recent work has shown that Toll-like receptor (TLR) ligation is an important signal affecting B cell activation. We sought to determine the effect that the combination of antigen recognition, T cell help and TLR ligation would have on B cell response profiles of both control donors and Multiple Sclerosis (MS) patients. We found that TLR8 ligation resulted in enhanced B cell proliferation and survival combined with a marked suppression of B cell effector cytokines. TLR9 ligation resulted in similar enhancement in B cell proliferation that was combined with significantly increased effector cytokine production. These findings highlight the importance of TLR ligation on B cell activation and underscore the importance of understanding the effects of TLR activation on B cell biology.Les cellules B deviennent de plus en plus reconnues comme étant des cellules immunorégulatrices importantes. Selon leur contexte d'activation, l'équilibre entre les cytokines pro-inflammatoires et anti-inflammatoires produites par les cellules B est affecté. Les travaux récents ont prouvé que l'activation des récepteurs "Toll-like" (communément appelés Toll-like receptors ou TLR) est un signal important affectant l'activation des cellules B. Nous avons cherché à determiner si l'activation des TLR combinée à l'identification d'antigène et l'aide des cellules T pouvait influencer les profils « effecteurs » des cellules B provenant de donneurs contrôles et de patients atteints de la sclérose en plaques (SP). Nous avons constaté que l'activation de TLR8 induit une augmentation de la prolifération et de la survie des cellules B, associée à une suppression marquée des cytokines effectrices sécrétées par les cellules B. L'activation de TLR9 a également induit une augmentation de la prolifération des cellules B, associée à une production sensiblement accrue de cytokines effectrices. Ces résultats accentuent l'important de l'activation des TLR sur l'activation des cellules B et soulignet l'importance de comprendre les effets de l'activation de TLR sur la biologies des cellules B

Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates

Thrombocytes, nucleated hemostatic blood cells of nonmammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5–3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions

Evolutionary conservation of divergent pro-inflammatory and homeostatic responses in Lamprey phagocytes.

In higher vertebrates, phagocytosis plays a critical role in development and immunity, based on the internalization and removal of apoptotic cells and invading pathogens, respectively. Previous studies describe the effective uptake of these particles by lower vertebrate and invertebrate phagocytes, and identify important molecular players that contribute to this internalization. However, it remains unclear if individual phagocytes mediate internalization processes in these ancient organisms, and how this impacts the balance of pro-inflammatory and homeostatic events within their infection sites. Herein we show that individual phagocytes of the jawless vertebrate Petromyzon marinus (sea lamprey), like those of teleost fish and mice, display the capacity for divergent pro-inflammatory and homeostatic responses following internalization of zymosan and apoptotic cells, respectively. Professional phagocytes (macrophages, monocytes, neutrophils) were the primary contributors to the internalization of pro-inflammatory particles among goldfish (C. auratus) and lamprey (P. marinus) hematopoietic leukocytes. However, goldfish showed a greater ability for zymosan phagocytosis when compared to their jawless counterparts. Coupled to this increase was a significantly lower sensitivity of goldfish phagocytes to homeostatic signals derived from apoptotic cell internalization. Together, this translated into a significantly greater capacity for induction of antimicrobial respiratory burst responses compared to lamprey phagocytes, but also a decreased efficacy in apoptotic cell-driven leukocyte homeostatic mechanisms that attenuate this pro-inflammatory process. Overall, our results show the long-standing evolutionary contribution of intrinsic phagocyte mechanisms for the control of inflammation, and illustrate one effective evolutionary strategy for increased responsiveness against invading pathogens. In addition, they highlight the need for development of complementary regulatory mechanisms of inflammation to ensure continued maintenance of host integrity amidst increasing challenges from invading pathogens

In-Cell Labeling Coupled to Direct Analysis of Extracellular Vesicles in the Conditioned Medium to Study Extracellular Vesicles Secretion with Minimum Sample Processing and Particle Loss

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture

Phagocytosis of different target particles by goldfish and lamprey primary leukocytes.

<p>(A) Goldfish primary kidney leukocytes (PKL) or lamprey primary typhlosole leukocytes (PTL) were incubated with 3 µm YG latex beads, <i>E. coli</i> DH5α-GFP, or zymosan-FITC at the indicated concentrations for the specified times. Cells were then fixed and phagocytosis was quantified by flow cytometry. Grey bars represent percent internalized. Hatched white bars represent percent surface bound. For all n = 4, over 2 examined over a minimum of two independent experiments. * p<0.05 for % internalized,+p<0.05 for % surface bound- between 10∶1 and 5∶1 particle to cell ratios in each graph. (B) Representative images of no internalization (No), surface bound (B), and internalized beads (Int.) from ImageStream MkII flow cytometer (Amnis).</p

Effect of pre-incubation with zymosan and apoptotic cells on respiratory burst responses of individual phagocytes.

<p>Goldfish PKL and lamprey PTL were incubated with both zymosan and apoptotic cells (5∶1 ratio for each) for 2 h and 6 h, respectively. (A) To investigate the effects of pre-incubation with apoptotic cells, apoptotic cells were added 2 h prior to zymosan. Respiratory burst (measured as % DHR positive) was then analyzed based on phagocytic capacity across the four resulting sub-populations: non-phagocytic cells, phagocytes containing only zymosan, phagocytes containing only apoptotic cells, and phagocytes that contain both. (B) The percent of total population found in each of the four sub-populations of (A); no internalization, zymosan only, apoptotic cells only, zymosan and apoptotic cells. (C) The phagocytic index of the Zym+AC group in (A). For all n = 4, examined over a minimum of two independent experiments. * p<0.05 and ** p<0.01 compared to No;+p<0.05 and++p<0.01 compared to Zym.</p