A flow cytometry method for safe detection of bacterial viability.

Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris-Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non-CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability-DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram+ , Gram- ) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization.

Picolylamine-functionalized benz[e]indole squaraine dyes: Synthetic approach, characterization and in vitro efficacy as potential anticancer phototherapeutic agents.

Squaraine dyes are a family of compounds known for their relevant photophysical and photochemical properties potentially useful as photosensitizing agents. Since pyridines have been introduced into the skeleton of several families of compounds to enhance their pharmacological activity, and this approach had not yet been performed on squaraines, novel dyes derived from benz[e]indole functionalized with picolyl- and dipicolylamine and N-ethyl and -hexyl chains were designed and synthesized. After being fully characterized, their interaction with human albumin was in vitro and in silico evaluated. Dyes were further assessed for their phototoxicity activity, and the most interesting ones were studied regarding cell localization and induction of morphological cell changes, genotoxicity, apoptosis and cell cycle arrest. The molecules with N-ethyl chains showed the greatest in vitro light-dependent cytotoxic effects, particularly the zwitterionic squaraine dye and the one bearing a single pyridine unit, which also exhibited a more significant interaction with human albumin. Phenotypically, the cells incubated with these squaraines became smaller and rounded after irradiation, the effects varying with the tested concentration. Genotoxic effects were observed even without irradiation, being more evident for the N-ethyl picolylamine-derived dye. The fluorescence emitted by Rhodamine 123 largely coincided with that emitted by the dyes, suggesting that they are found preferentially in mitochondria. After irradiation, an increase in the subG1 population was verified by propidium iodide-staining analysis by flow cytometry, indicative of cell death by apoptosis.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells.

BACKGROUND: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. METHODS: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. RESULTS: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. CONCLUSIONS: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

An Annexin V-FITC-Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line.

Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.

Assays of Eosinophil Apoptosis and Phagocytic Uptake.

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure of phosphatidylserine on the cell membrane. A greater understanding of apoptotic mechanisms, subsequent phagocytosis (efferocytosis), and regulation of these processes is critical to understanding disease pathogenesis and development of potential novel therapeutic agents. Release of soluble factors and alterations to surface marker expression by eosinophils undergoing apoptosis aid them in signaling their presence to the immediate environment, and their subsequent recognition by phagocytic cells such as macrophages. Uptake of apoptotic cells usually suppresses inflammation by restricting proinflammatory responses and promoting anti-inflammatory and tissue repair responses. This, in turn, promotes resolution of inflammation. Defects in the apoptotic or efferocytosis mechanisms perpetuate inflammation, resulting in chronic inflammation and enhanced disease severity. This can be due to increased eosinophil life span or cell necrosis characterized by loss of cell membrane integrity and release of toxic intracellular mediators. In this chapter, we detail some of the key assays that are used to assess eosinophil apoptosis, as well as the intracellular signaling pathways involved and phagocytic clearance of these cells.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1-5 µs × 250 and 100 µs × 1-8 pulsing protocols in 1-2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

Ultrathin glass fiber microprobe for electroporation of arbitrary selected cell groups.

A new type of ultrathin fiber microprobe for selective electroporation is reported. The microprobe is 10 cm long and has a diameter of 350 µm. This microprobe is a low cost tool, which allows electroporation of an arbitrary selected single cell or groups of cells among population with use of a standard microscope and cell culture plates. The microprobe in its basic form contains two metal microelectrodes made of a silver-copper alloy, running along the fiber, each with a diameter of 23 µm. The probe was tested in vitro on a population of normal and cancer cells. Successful targeted electroporation was observed by means of accumulation of trypan blue (TB) dye marker in the cell. The electroporation phenomenon was also verified with propidium iodide and AnnexinV in fluorescent microscopy.

Emodin Induces Death in Human Cervical Cancer Cells Through Mitotic Catastrophe.

BACKGROUND: Anthraquinones, including emodin, are compounds with numerous pharmacological properties, including anticancer properties. The aim of this study experiment was to examine the effect of emodin, a natural compound present in the roots and rhizomes of Rheum palmatum, on the induction of mitotic catastrophe in cervical cancer cells. MATERIAL AND METHODS: HeLa celIs were treated with different emodin concentrations for 48 h, and cell growth was measured with 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolyl. The cell-cycle distribution and the level of apoptosis were determined by means of flow cytometry, using annexin V-fluorescein isothiocyanate staining and propidium iodide. Morphological changes in the mitotic apparatus were evaluated using optical and confocal microscopy techniques. RESULTS: Emodin induced an increase in the number of polymorphonuclear cells, giant cells, cells with micronuclei, cells with abnormal mitosis and damaged spindle. The reorganization of F-actin depended on the concentration of emodin. With the increase in emodin concentration, inhibition of mitotic activity was demonstrated, which was manifested by a decrease in the mitotic index, mainly in metaphase of the mitotic process and an increase in the number of cells inhibited in the G2/M phase. At the same time, an increase in the number of apoptotic cells was found. CONCLUSION: Emodin leads to death of cervical cancer cells by induction of a mitotic catastrophe.

Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells.

OBJECTIVE: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS: The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.

Cell Harvesting Methods Affect Cellular Integrity of Adherent Cells During Apoptosis Detection.

BACKGROUND/AIM: Annexin V and propidium iodide (PI) dual staining is commonly applied in bioscience as a method to detect apoptosis. However, excessive handling of adherent cells may interfere with the integrity of plasma membrane and hence impede the accuracy of this method. Here, we exploited PI uptake as an indicator of cell integrity and investigated how cell harvesting methods and solutions involved in common apoptosis detection techniques affected measurement results. MATERIALS AND METHODS: Different cell harvesting techniques, staining with PI and flow cytometry were performed. RESULTS: Non-fixed scrapped cells revealed significantly higher fractions of PI-positive staining compared to non-fixed trypsinized cells. In the case of harvesting cells by scrapping, samples stained in binding buffer (68.30±3.55%) showed consistently higher PI-positive staining than samples stained in PBS (36.37±5.90%) in a significant manner (p=0.015). CONCLUSION: Enzymatic harvesting using 0.25% trypsin instead of mechanical harvesting by rubber scraper caused less damage of cell integrity. Furthermore, the binding buffer used in the apoptosis detection protocol aggravated the existing plasma membrane damage caused by the rubber scraper.

Antrodia cinnamomea boosts the anti-tumor activity of sorafenib in xenograft models of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.

Cellular fluorescein hyperfluorescence is dynamin-dependent and increased by Tetronic 1107 treatment.

Sodium fluorescein ('fluorescein') staining of the ocular surface is frequently an indicator of compromised ocular health, and increases in the presence of certain contact lens multi-purpose solutions (MPS), a phenomenon known as solution induced corneal staining (SICS). The mechanism(s) underpinning fluorescein hyperfluorescence are uncertain, though may reflect increased cellular uptake of fluorescein by corneal epithelial cells. We have developed an in vitro model to study fluorescein uptake in both 'generic' mammalian cells (murine fibroblasts) and human corneal cells. Fluorescein hyperfluorescence increased after treatment with two MPS associated with clinical corneal fluorescein staining, yet there was no cellular hyperfluorescence for two MPS that do not cause this staining. Increased fluorescein uptake did not correlate with presence of a necrotic or an apoptotic marker (propidium iodide and caspase-3 respectively). Incubation of MPS-treated cells with dynasore (an inhibitor of dynamin, implicated in endocytic pathways) reduced fluorescein uptake irrespective of MPS treatment. The non-ionic surfactant Tetronic 1107 (present in both MPS associated with corneal fluorescein staining) increased uptake of fluorescein for both cell types, whereas an unrelated surfactant (Triton X-100) did not. We conclude that the clinical hyperfluorescence profile observed after exposure to four MPS can be reproduced using a simple model of cellular fluorescein uptake, suggesting this is the biological basis for SICS. Fluorescein entry does not correlate with necrosis or apoptosis, but instead involves a dynamin-dependent active process. Moreover the surfactant Tetronic 1107 appears to be a key MPS constituent triggering increased fluorescein entry, and may be the major factor responsible for SICS.

Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation.

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G0/G1, sub-S and sub-G2/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Effects of drinking desalinated seawater on cell viability and proliferation.

Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

Ferroptosis and Cell Death Analysis by Flow Cytometry.

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

Cell Viability of Human Embryonic Stem Cells Stored for a Period of 9 Years.

OBJECTIVES: Human embryonic stem cells are pluripotent cell lines usually derived from human blastocysts. Their potential critically depends on long-term proliferative capacity, developmental potential after prolonged culture, and karyotypic stability. Cell viability is an important parameter for assessing cell sample quality. Here, we elaborate the stored human embryonic stem cell lines' viability in a ready to use form for a period of 9 years (from 2007 to 2015). MATERIALS AND METHODS: Spare pre implantation stage in vitro fertilized ovum-derived cell lines were cultured in suitable media. Thereafter, they were centrifuged at 1000 revolutions/min over 5 minutes, and pellets were suspended in normal saline. Next, they were tested for viability from storage at -20°C. After being allowed to thaw slowly, the cells were stained with propidium iodide and analyzed using flow cytometry. Images of cells were taken at ×40 and ×100 magnification. RESULTS: At ×100 magnification, cell population size ranged from 0.2 to 2 µm. The percentage of live cells was more than 95% throughout the 9 years. Cells frozen in 2015 showed cell viability of 96.8%. CONCLUSIONS: We observed high cell viability in our cell lines for 9 years. Human embryonic stem cell lines in a ready-to-use form can be preserved for long-term purposes. Thus, they could be made available globally.

Peptoid Efficacy against Polymicrobial Biofilms Determined by Using Propidium Monoazide-Modified Quantitative PCR.

Biofilms containing Candida albicans are responsible for a wide variety of clinical infections. The protective effects of the biofilm matrix, the low metabolic activity of microorganisms within a biofilm and their high mutation rate, significantly enhance the resistance of biofilms to conventional antimicrobial treatments. Peptoids are peptide-mimics that share many features of host defence antimicrobial peptides but have increased resistance to proteases and therefore have better stability in vivo. The activity of a library of peptoids was tested against monospecies and polymicrobial bacterial/fungal biofilms. Selected peptoids showed significant bactericidal and fungicidal activity against the polymicrobial biofilms. This coupled with low cytotoxicity suggests that peptoids could offer a new option for the treatment of clinically relevant polymicrobial infections.