Establishment of a new method to isolate viable x-ray-sensitive cells from planarian by fluorescence-activated cell sorting.

Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence-activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x-ray-irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x-ray-irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x-ray-sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into x-ray-irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.

Nuclear receptor coactivator 4-mediated ferritinophagy contributes to cerebral ischemia-induced ferroptosis in ischemic stroke.

Ischemic stroke poses a significant health risk due to its high rate of disability and mortality. To address this problem, several therapeutic approaches have been proposed, including interruption targeting programmed cell death (PCD). Ferroptosis is a newly defined PCD characterized by iron-dependent accumulation of lipid peroxidation, and is becoming a promising target for treating numerous diseases. To explore the underlying mechanisms of the initiation and execution of ferroptosis in ischemic stroke, we established stroke models in vivo and in vitro simulating ischemia/reperfusion (I/R) neuronal injury. Different from previous reports on stroke, we tested ferroptosis by measuring the levels of core proteins, such as ACSL4, 15-LOX2, Ferritin and GPX4. In addition, I/R injury induces excessive degradation of ferritin via the autophagy pathway and subsequent increase of free iron in neurons. This phenomenon has recently been termed ferritinophagy and reported to be regulated by nuclear receptor coactivator 4 (NCOA4) in some cell lines. Increased NCOA4 in cytoplasm was detected in our study and then silenced by shRNA to investigate its function. Both in vivo and in vitro, NCOA4 deletion notably abrogated ferritinophagy caused by I/R injury and thus inhibited ferroptosis. Furthermore, we found that NCOA4 was upregulated by ubiquitin specific peptidase 14 (USP14) via a deubiquitination process in damaged neurons, and we found evidence of pharmacological inhibition of USP14 effectively reducing NCOA4 levels to protect neurons from ferritinophagy-mediated ferroptosis. These findings suggest a novel and effective target for treating ischemic stroke.

Exogenic production of bioactive filamentous biopolymer by monogonant rotifers.

The chemical ecology of rotifers has been little studied. A yet unknown property is presented within some monogonant rotifers, namely the ability to produce an exogenic filamentous biopolymer, named 'Rotimer'. This rotifer-specific viscoelastic fiber was observed in six different freshwater monogonants (Euchlanis dilatata, Lecane bulla, Lepadella patella, Itura aurita, Colurella adriatica and Trichocerca iernis) in exception of four species. Induction of Rotimer secretion can only be achieved by mechanically irritating rotifer ciliate with administering different types (yeast cell skeleton, denatured BSA, epoxy, Carmine or urea crystals and micro-cellulose) and sizes (approx. from 2.5 to 50 µm diameter) of inert particles, as inductors or visualization by adhering particles. The thickness of this Rotimer is 33 ± 3 nm, detected by scanning electron microscope. This material has two structural formations (fiber or gluelike) in nano dimension. The existence of the novel adherent natural product becomes visible by forming a 'Rotimer-Inductor Conglomerate' (RIC) web structure within a few minutes. The RIC-producing capacity of animals, depends on viability, is significantly modified according to physiological- (depletion), drug- (toxin or stimulator) and environmental (temperature, salt content and pH) effects. The E. dilatata-produced RIC is affected by protein disruptors but is resistant to several chemical influences and its Rotimer component has an overwhelming cell (algae, yeast and human neuroblastoma) motility inhibitory effect, associated with low toxicity. This biopolymer-secretion-capacity is protective of rotifers against human-type beta-amyloid aggregates.

Nisin penetration and efficacy against Staphylococcus aureus biofilms under continuous-flow conditions.

Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus. Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.

5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is often associated with overexpression of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2). Modulators of these transporters might be helpful in overcoming MDR. Moreover, exploiting collateral sensitivity (CS) could be another approach for efficient treatment of cancer. Twelve novel 5-oxo-hexahydroquinoline derivatives bearing different aromatic substitutions at C4, while having 2-pyridyl alkyl carboxylate substituents at the C3 were synthesized and evaluated for MDR reversal activity by flow cytometric determination of rhodamine 123, calcein and mitoxantrone accumulations in P-gp, MRP1 and BCRP-overexpressing cell lines, respectively. Furthermore, to confirm the P-gp inhibitory activity, the effect of compounds on the reduction of doxorubicin's IC50 of drug-resistant human uterine sarcoma cell line, MES-SA/DX5, was evaluated. Compounds D6, D5 and D3 (bearing 3-chlorophenyl, 2,3-dichlorophenyl and 4-chlorophenyl substituents at C4 position of 5-oxo-hexahydroquinoline core) were the most potent P-gp, MRP1 and BCRP inhibitors, respectively, causing significant MDR reversal at concentrations of 1-10 µM. Additionally, D4 (containing 3-flourophenyl) was the most effective MRP1-dependent CS inducing agent. Overall, chlorine containing compounds D6, C4 and D3 were capable of significant inhibition of all 3 important efflux pumps in cancer cells. Moreover, D6 also induced CS triggered by reducing glutathione efflux. In conclusion, some of the 5-oxo-hexahydroquinoline derivatives are effective efflux pump inhibitors capable of simultaneously blocking 3 important ABC transporters involved in MDR, and represent promising agents to overcome MDR in cancer cells.

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia.

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = -0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by 14C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.

Long-term inhibition of cyclophilin D results in intracellular translocation of calcein AM from mitochondria to lysosomes.

Cyclophilin D is a peptidyl-prolyl cis-trans isomerase localized in the mitochondrial matrix. Although its effects on mitochondrial characteristics have been well studied, its relation to the uptake of molecules by mitochondria remains unknown. Here, we demonstrated the effects of cyclophilin D on the intracellular translocation of calcein AM. Following addition of calcein AM to control cells or cells overexpressing wild-type cyclophilin D, calcein fluorescence was observed in mitochondria. However, long-term inhibition of cyclophilin D in these cells altered the localization of calcein fluorescence from mitochondria to lysosomes without changing mitochondrial esterase activity. In addition, depletion of glucose from the medium recovered calcein localization from lysosomes to mitochondria. This is the first demonstration of the effects of cyclophilin D on the intracellular translocation of molecules other than proteins and suggests that cyclophilin D may modify mitochondrial features by inducing the translocation of molecules to the mitochondria through the mechanism associated with cellular energy metabolism.

Streamlined duplex live-dead microplate assay for cultured cells.

A duplex fluorescence assay to assess the viability of cells cultured in multi-well plates is described, which can be carried out in the original culture plate using a plate reader, without exchanges of culture or assay medium, or transfer of cells or cell supernatant. The method uses freshly prepared reagents and does not rely on a proprietary, commercially supplied kit. Following experimental treatment, calcein acetoxymethyl ester (CaAM) is added to each well of cultured cells; after 30 min, the fluorescence intensity (emission λmax âˆ¼ 530 nm) is measured. The signal is due to formation of calcein, which is produced from CaAM by action of esterase activity found in intact live cells. Since live cells may express plasma membrane multidrug transport proteins, especially of the ABC transporter family, the CaAM incubation is carried out in the presence of an inhibitor of this efflux process, thereby improving the dynamic range of the assay. Next, SYTOX® Orange (SO) is added to the culture wells, and, after a 30-min incubation, fluorescence intensity (emission λmax âˆ¼ 590 nm) is measured again. SO is excluded from cells that have an intact plasma membrane, but penetrates dead/dying cells and can diffuse into the nucleus, where it binds to and forms a fluorescent complex with DNA. The CaAM already added to the wells causes no interference with the latter fluorescent signal. At the conclusion of the duplex assay, both live and dead cells remain in the culture wells and can be documented by digital imaging to demonstrate correlation of cellular morphology with the assay output. Two examples of the application of this method are provided, using cytotoxic compounds having different mechanisms of action.

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.

A new and sensitive method for measuring in vivo and in vitro cytotoxicity in earthworm coelomocytes by flow cytometry.

This study describes a new and sensitive method for measuring the in vivo and in vitro cytotoxicity of 2 earthworm species, Eisenia andrei and Perionyx excavatus, exposed to copper. Specifically, we measured the number of coelomocyte cells that were affected by copper following in vivo and in vitro exposure by flow cytometry, after calcein acetoxymethyl ester (calcein-AM) staining. We found that the coelomocyte viability of both earthworm species was noticeably reduced in the in vivo cytotoxicity test at concentrations of 100mg/kg copper in dry soil. However, pathological symptoms, such as mucous secretion and bleeding, swelling, thinning, and fragmentation, and burrowing symptoms were not evident following exposure to copper levels of <400mg/kg dry soil. In conclusion, the present study demonstrates that calcein-AM is a more sensitive test of earthworm coelomocyte cytotoxicity compared to the traditional individual level toxicity test. Therefore, this test could be used to detect low levels of metal contamination in soils.

Inhibition of P-glycoprotein-mediated efflux by thiolated cyclodextrins.

Overcoming P-glycoprotein (P-gp)-mediated efflux poses a significant challenge for the pharmaceutical industry. This study investigates the potential of thiolated ß-cyclodextrins (ß-CD-SHs) as inhibitors of P-gp-mediated efflux in Caco-2 cells. Through a series of transport assays, intracellular accumulation, and efflux of the P-gp substrates Rhodamine 123 (Rh123) and Calcein-AM with and without co-administration of ß-CD-SHs were assessed. The results revealed that the cellular uptake of Rh123 and Calcein-AM were enhanced up to 7- and 3-fold, compared to the control, respectively. In efflux studies an up to 2.5-fold reduction of the Rh123 efflux was reached compared the control, indicating a substantial decrease of Rh123 efflux by ß-CD-SHs. Furthermore, it was observed that ß-CD-SHs led to a decrease in the reactivity of fluorescence-labeled anti-P-gp, suggesting additional effects on the conformation of P-gp. Overall, this study demonstrates the potential of ß-CD-SHs as effective modulator of P-gp-mediated drug efflux in Caco-2 cells.

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

BACKGROUND & AIMS: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.

Overexpression of a GIPC glycosyltransferase gene, OsGMT1, suppresses plant immunity and delays heading time in rice.

Glycosylinositol phosphorylceramides (GIPCs) are the major sphingolipids in the plant plasma membrane. In Arabidopsis, mutations of genes involved in the synthesis of GIPCs affect many physiological aspects of plants, including growth, pollen fertility, defense, and stress signaling. Loss of function of the GIPC MANNOSYL-TRANSFERASE1 (AtGMT1) results in GIPC misglycosylation and induces plant immune responses accompanied by a severely dwarfed phenotype, thus indicating that GIPCs play important roles in plant immunity. Here, we investigated the enzymatic activity and phenotypes of transgenic lines of OsGMT1, the ortholog of AtGMT1. Sphingolipidomic analysis indicated that OsGMT1 retained the enzymatic activity of GIPC hexose (Hex) glycosylation, but the knockout lines did not accumulate H2O2. In contrast, the OsGMT1 overexpression lines showed significant down-regulation of several defense-associated or cell wall synthesis-associated genes, and enhanced sensitivity to rice blast. Furthermore, we first demonstrated the sensitivity of rice cells to MoNLP1 protein through calcein AM release assays using rice protoplasts, thus legitimizing the presence of MoNLPs in rice blast fungus. In addition, yeast two-hybrid screens using OsGMT1 as bait revealed that OsGMT1 may regulate heading time through the OsHAP5C signaling pathway. Together, our findings suggested clear physiological functional differentiation of GMT1 orthologs between rice and Arabidopsis.

Drug-free and non-crosslinked chitosan/hyaluronic acid hybrid hydrogel for synergistic healing of infected diabetic wounds.

The management of infected diabetic wounds remains a major challenge in clinical practice. Recently, multifunctional hydrogels have attracted much attention in the area of wound healing. Herein, we developed the drug-free and non-crosslinked chitosan (CS)/hyaluronic acid (HA) hybrid hydrogel, so as to combine the multiple functions of CS and HA for synergistic healing of the methicillin-resistant Staphylococcus aureus (MRSA)-infected diabetic wound. As a result, CS/HA hydrogel showed the broad-spectrum antibacterial activity, the great capacity for promoting fibroblasts proliferation and migration, the excellent reactive oxygen species (ROS) scavenging ability, and the great cell-protection effects under oxidative stress. In the MRSA-infected diabetic mouse wounds, CS/HA hydrogel significantly promoted the wound healing via eliminating MRSA infection and enhancing epidermal regeneration, collagen deposition and angiogenesis. Considering the drug-free feature, the ready availability, the great biocompatibility and the excellent wound healing efficacy, CS/HA hydrogel may have great potentials in clinical use for the management of chronic diabetic wounds.

Carboxymethyl chitosan-assisted MnOx nanoparticles: Synthesis, characterization, detection and cartilage repair in early osteoarthritis.

While the early detection and repair of cartilage lesions are crucial in the treatment of osteoarthritis (OA), they remain challenging because neither clinically used medicines nor magnetic resonance (MR) contrast agents can achieve detection and repair simultaneously. Here, we conjugated carboxymethyl chitosan (CMC) with a cartilage-targeting peptide (WYRGRL, termed WY) and then synthesized CMC-assisted manganese oxide nanoparticles (MnOx NPs). The resultant WY-CMC-MnOx NPs demonstrated an excellent biocompatibility and a good T1 relaxivity of 1.72 mM-1·s-1. Owing to their ultrasmall size and cartilage-targeting ability, the WY-CMC-MnOx NPs considerably increased the MR imaging quality of cartilage lesions compared to non-cartilage-targeting NPs. In contrast, clinically used gadolinium-diethylenetriamine pentaacetic acid (Gd-DPTA) failed to detect the cartilage lesions. Furthermore, WY-CMC-MnOx promoted chondrogenesis in mesenchymal stem cells, thereby enhancing OA therapy through efficient cartilage regeneration after intraarticularly injection in destabilization of medial meniscus (DMM) rat models. Our results indicate that WY-CMC-MnOx NPs are promising for use in the diagnosis and treatment of early OA.

FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1.

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H2 DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4 (3)] and SYTOX-Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold-water, 26 temperate, and four warm-water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein-AM and H2 DCFDA (P < 0.001). Of the two membrane probes, DIBAC4 (3) stained rhodophytes and euglenophytes much better than SYTOX-Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC4 (3), and SYTOX-Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.

Interaction of Commonly Used Oral Molecular Excipients with P-glycoprotein.

P-glycoprotein (P-gp) plays a critical role in drug oral bioavailability, and modulation of this transporter can alter the safety and/or efficacy profile of substrate drugs. Individual oral molecular excipients that inhibit P-gp function have been considered a mechanism for improving drug absorption, but a systematic evaluation of the interaction of excipients with P-gp is critical for informed selection of optimal formulations of proprietary and generic drug products. A library of 123 oral molecular excipients was screened for their ability to inhibit P-gp in two orthogonal cell-based assays. ß-Cyclodextrin and light green SF yellowish were identified as modest inhibitors of P-gp with IC50 values of 168 µM (95% CI, 118-251 µM) and 204 µM (95% CI, 5.9-1745 µM), respectively. The lack of effect of most of the tested excipients on P-gp transport provides a wide selection of excipients for inclusion in oral formulations with minimal risk of influencing the oral bioavailability of P-gp substrates.

Measurement of canine blood microparticles by flow cytometry: effect of anticoagulants and staining reagents.

Microparticles (MPs) are released from budding plasma membranes into body fluids. The use of flow cytometry for the measurement of MP in canines has not been standardized. In this fundamental study, we compared the effect of anticoagulant agents, such as acid-citrate-dextrose (ACD) and heparin on the measurement of canine MPs in platelet-free plasma (PFP) using flow cytometry. In addition, we used annexin V, carboxyfluorescein succinimidyl ester (CFSE), or calcein tetraacetoxymethyl ester (calcein-AM), and explored the characteristics of the staining reagents in MP detection using flow cytometry. We were able to measure canine MPs in PFP prepared from ACD-anticoagulated blood using flow cytometry, in which the highest positive rate for fluorescent staining was observed when CFSE was used.

Comparative Evaluation of Natural Killer Cell-mediated Cell Killing Assay Based on the Leakage of an Endogenous Enzyme or a Pre-loaded Fluorophore.

A cell-killing ability of natural killer (NK) cells has been evaluated by the leakage of marker molecules from target cells. Lactate dehydrogenase (LDH) and calcein are two major non-radioisotope markers used for the killing assay. The spontaneous death of NK cells during the killing-assay of cells is a major issue in the assay because it provides background signals to increase errors. In this study, the effect of the spontaneous death of NK cells on the killing assays based on LDH and the calcein method was comparatively evaluated. We found that the calcein method is much less sensitive to the spontaneous death of NK cells to enable an accurate evaluation of the cell killing.