Flow-Cytometer

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EVALUATION OF ANTIMICROBIAL STUDIES ON ETHANOLIC EXTRACTS AND POLY HERBAL FORMULATIONS OF
ABUTILON INDICUM, ARISTOLOCHEA BRACTEOLATE, AND ANDROGRAPHIS PANICULATA BY USING FLOW CYTOMETER

EVALUATION OF ANTIMICROBIAL STUDIES ON ETHANOLIC EXTRACTS AND POLY HERBAL FORMULATIONS OF ABUTILON INDICUM, ARISTOLOCHEA BRACTEOLATE, AND ANDROGRAPHIS PANICULATA BY USING FLOW CYTOMETER

The samples were run on low mode. Individual cells were detected by their forward-angle (FSC) and right-angle scatter (SSC) of incident 488-nm laser light. This light scatter data plot was used to establish a gated region that excluded cell clusters and debris from the fluorescence analysis. PI fluorescence of individual cells was acquired by using a FL3 detector (Em = 630-nm) and displayed in dot plots. Flow cytometer’s onboard software (Cell Quest Pro), was used to analyze the fluorescence dot plots and to determine the percentage of PI - (living) and PI + (dead) bacterial cells.
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Evaluation of the Partec Flow Cytometer against the BD FACSCalibur System for Monitoring Immune Responses of Human Immunodeficiency Virus-Infected Patients in Zimbabwe

Evaluation of the Partec Flow Cytometer against the BD FACSCalibur System for Monitoring Immune Responses of Human Immunodeficiency Virus-Infected Patients in Zimbabwe

Equipment used in the study. (i) Partec Cyflow SL_3. The Partec Cyflow SL_3 is a single-platform, three-parameter (SSC plus two-color fluorescence) desktop flow cytometer. It contains a solid-state laser for green excitation. It analyzes concentrations of any particle or cell subpopulation of interest, using true volu- metric absolute counting. For data analysis, the analyzer uses Flomax software. The Partec CD4% Reagent kit used contained direct immunofluorescence re- agents for enumeration of mature CD4 ⫹ T lymphocytes and, simultaneously, of CD45 ⫹ cells in peripheral blood. The kit consists of a monoclonal antibody, MEM-241, which recognizes the human CD4 antigen, a transmembrane glyco- protein (59 kDa) of the immunoglobulin supergene family, present on a subset of T lymphocytes (“helper/inducer” T cells) and expressed at lower levels on monocytes and granulocytes. Approximately 20 to 60% of human peripheral blood mononuclear cells and a subpopulation of monocytes were stained, albeit TABLE 1. Raw data for method precision, with measurements by the Partec Cyflow SL_3 and the BD FACSCalibur/Sysmex XT1800i
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Flow Cytometer Performance Characterization, Standardization and Calibration against CD4 on T Lymphocytes Enables Quantification of Biomarker Expressions for Immunological Applications

Flow Cytometer Performance Characterization, Standardization and Calibration against CD4 on T Lymphocytes Enables Quantification of Biomarker Expressions for Immunological Applications

Quantitative flow cytometry has been demonstrated in a single laboratory study [9]-[11]. However, multicen- ter quantitative studies including various flow cytometer platforms have not yet been reported, suggesting the enormous difficulty of the task. This type of study requires both multi-platform instrument standardization and the use of a biological reference marker with a known expression level for ultimate biomarker quantification. The instrument standardization includes multiple steps, e.g. characterization of each instrument performance, target instrument identification, optimization of the target instrument fluorescence detector voltages as well as the application of the median fluorescence intensity (MFI) values from the target instrument to other instrument platforms. Once all flow cytometers are standardized, cell biomarker expression levels can be quantified with a reference biomarker, e.g. CD4 on normal human T lymphocytes with a known number of the antibody bound per cell (ABC) value [12]-[14]. In the present study, we have developed an instrument standardization procedure across three different flow cytometer platforms from the same vendor with somewhat different optical configu- rations and in two different locations. After the standardization of these instruments, CD19 quantification is car- ried out to validate the ABC quantification approach and demonstrate that consistent and reliable results can be accomplished between instruments using the developed procedure.
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Automated bedside flow cytometer for mHLA-DR expression measurement: a comparison study with reference protocol

Automated bedside flow cytometer for mHLA-DR expression measurement: a comparison study with reference protocol

Overall, at this stage, except for this aberrant value out of 139 measurements, this first beta-site Accellix study regarding mHLA-DR assessment is very encouraging. Al- though we had no access to the algorithm and flow histograms, we noticed satisfactory results for a comparison study. In addition, despite known pre-analytical issues [9], re- peatability results were found to be within acceptable range for flow cytometry. To date, mHLA-DR, although providing excellent information, has remained under- utilized due to the difficulty to use/have access to flow cytometry. In contrast, Accellix may provide a workable solution for this problem. The sole human intervention is to deposit about 40 μL of whole blood on the cartridge. As there is no flow histogram available on Accellix, there is no interpretation of the results based on usual gating strategy or fluorescence compensation. No specific technical skills in flow cytometry are thus required. Accellix provides a result as mHLA-DR index—nothing else. Thus, Accellix may be installed directly in ICUs or in 24/7 emergency labs where traditional flow cytometer are not present. Beyond routine monitoring, in the short term, it may facilitate managing multicentered clinical trials based on mHLA-DR patient stratifica- tion. To date, the main Accellix drawback lies in the fact that it can only analyze a sin- gle cartridge at a time and requires 25 min to do so. This implies a need to correctly schedule blood sampling and analysis onset when multiple samples are to be analyzed simultaneously as blood cannot be stored for too long before mHLA-DR staining. By nature, in a beta site evaluation, clinical decision-making thresholds are not investi- gated. Thus, a next important step would be to conduct a comparative study in a larger cohort of patients to define and assess those thresholds in predicting mortality and/or secondary infection occurrence and to evaluate how they interface with usual values from HLA-DR as ABC obtained with standard protocol.
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Screening for Urinary Tract Infection with the Sysmex UF 1000i Urine Flow Cytometer

Screening for Urinary Tract Infection with the Sysmex UF 1000i Urine Flow Cytometer

bacteria and white blood cells, are fast but lack sensitivity (5, 21). Moreover, microscopic sediment analysis suffers from in- terobserver variation and is also labor-intensive. Automated methods for urine sediment analysis, like the Sysmex UF-100, have been developed. The Sysmex UF-100 is an automated urine flow cytometer able to detect particles in urine, including leukocytes and bacteria, quickly by staining the particles with fluorescent dyes and with subsequent identification by imped- ance, scattering, and fluorescence. Several groups have com- pared the results of the Sysmex UF-100 to microscopic sedi- ment analysis and reported an adequate performance of the analyzer (3, 7, 17, 18). However, the results of studies that compare the bacterial and leukocyte counts with the UF-100 with urine culture for the diagnosis of UTI vary widely. While some studies have reported an adequate performance in rela- tion to urine culture (6, 9, 10, 13), others have found a rela- tively large number of false negatives, which decreases sensi- tivity and makes the UF-100 unsuitable as a screening method to detect negative culture samples (2, 15, 22). Comparing stud- ies is difficult though, since reported sensitivities and specific- ities depend on the definition used for gold standard positive and negative urines, and these definitions vary among labora- tories. For example, some authors consider a urine sample positive if it contains more than 10 3 CFU/ml (2, 9), whereas
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Evaluation of a method for counting absolute numbers of cells with a flow cytometer.

Evaluation of a method for counting absolute numbers of cells with a flow cytometer.

measured 55 instead of 50 ml of whole blood, this could explain the increased values. The pipettes used in this study, however, were carefully calibrated by the manufacturer to deliver 50 ml. Another explanation might be that the hematology instru- ments were measuring inaccurately. That seems unlikely since, though they are located in different laboratories, they per- formed similarly and the quality control for both instruments was carefully monitored. Finally, the method for measuring lymphocytes in a hematology analyzer is considerably different from that for TruCount. TruCount tubes containing a known number of beads are provided by the manufacturer. While any factors that affect the number of beads in the tubes could have an impact on the final result, we assume that the beads are provided in the numbers indicated on the tubes. Also, the TruCount system is a no-wash system, so cell loss is minimized, and calibrator beads are run on a daily basis to ensure that the flow cytometer is counting correctly. Thus, we are unable to explain why we obtained higher counts with TruCount than with conventional methods.
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P53 Expression in Acute Lymphblastic Leukemia in Sudanese Patients Using Flow Cytometer

P53 Expression in Acute Lymphblastic Leukemia in Sudanese Patients Using Flow Cytometer

This study was conducted to detect of p53 expression in acute lymphocytic leukemia in Sudanese patients using the FlowCytometry and data were collected from the pa[r]

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Development of a helium neon laser based flow cytometer for evaluation of particulate matter

Development of a helium neon laser based flow cytometer for evaluation of particulate matter

SYSTEM DESCRIPTION Overview The most crucial part of the system is the flow chamber which is used to confine cells in the central part of the flow stream for exposure to the laser beam w[r]

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Original Article Effects of target regulation of LDHA through the PDK1/Akt/mTOR pathway on myocardial apoptosis caused by ischemia/reperfusion injury in rats

Original Article Effects of target regulation of LDHA through the PDK1/Akt/mTOR pathway on myocardial apoptosis caused by ischemia/reperfusion injury in rats

Abstract: Objective: To explore the effect and mechanism of lactate dehydrogenase A (LDHA) on myocardial apop- tosis in rats with myocardial ischemia-reperfusion injury (MIRI). Methods: Thirty newborn Wistar rats were killed and dissected under aseptic condition, then their hearts were cut for primary culture of cardiomyocytes. A model of myocardial hypoxia/reoxygenation (H/R) injury was established in vitro. The mRNA and protein expression levels of LDHA in rats’ primary cardiomyocytes were tested according to Real-time PCR and Western blot methods. LDHA overexpression vectors were constructed and transfected into cardiomyocytes, and were divided into 4 groups: control group, control +LDHA transfection group, H/R group and H/R+LDHA transfection group. CCK-8 method was used to detect myocardial proliferation activity in each group, flow cytometer to detect myocardial apoptosis, Western blot method to detect the effect of the LDHA overexpression on PDK1/AKt/mTOR protein expression un- der H/R and dual-luciferase method to identify target regulation of LDHA to PDK1. Results: Compared with control group, the proliferation activity of rats’ primary cardiacmyocytes in H/R group reduced significantly (P=0.002), while the myocardial apoptosis and the expression of LDHA increased (P=0.001, P=0.000). Compared with H/R group, the overexpression of LDHA in H/R+LDHA transfection group could promote myocardial proliferation significantly (P=0.000), and inhibit apoptosis (P=0.001). And the results of Western blot suggested the expressions of AKt, mTOR and PDK1 proteins in H/R+LDHA transfection group increased significantly and the differences had statistical significance (P=0.000). Compared with control group, the results of dual-luciferase method showed the luciferase signal intensity of vectors carrying wild type PDK1 reporter gene decreased by 45%, but there was no significant changes in the luciferase signal intensity of vectors carrying mutant type PDK1 reporter gene. Conclusion: The in- creasing LDHA expression in cardiomyocytes could up-regulate the activity of PDK1/AKt/mTOR pathway, and inhibit myocardial apoptosis in H/R.
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Validation of a single platform, volumetric, flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit

Validation of a single platform, volumetric, flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit

Background: A mobile health unit may be useful to follow up adult and pediatric patients on antiretroviral treatment and living in remote areas devoid of laboratory facilities. The study evaluated the use of the simplified, robust, single-plateform, volumetric, pan-leucogating Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) for CD4 T cell numeration in a mobile unit, compared against a reference flow cytometry method. Methods: The therapeutic mobile unit of the Laboratoire National de Santé Hygiène Mobile, Yaoundé, Cameroon, was equipped with the Auto40. A FACSCalibur flow cytometer (Becton Dickinson Immuno-cytometry System, San Jose, CA, USA) was used as reference method. EDTA-blood samples from volunteers were first subjected to CD4 T cell count in the mobile unit, and an aliquot was sent within 4 hours to Centre International de Référence Chantal Biya, Yaoundé, for FACSCalibur assay.
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A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

but are generally expensive [13]. In this work, we propose to use a benchtop Accuri flow cytometer as the basis for the design of an automated FC. This apparatus was recently tested on microbiological samples and led to reliable results [14]. In addition, fluid displacement is ensured by peristaltic pumps, facilitating the set-up of an interface with a bioreactor since no pressurization of the sample is needed. The development of previous systems was indeed impaired by the need to maintain pressure at the level of the sample unit [15-17]. Under this condition, FC can be easily interfaced to a bio- reactor by using additional peristaltic pumps operated by a microcontroller. Sample dilution and staining is carried out in line in the tubing between the FC and the bioreac- tor. This automated FC system was tested by following the dynamics of an Escherichia coli pfis::gfpAAV fluores- cent bio-reporter [18]. The reporter system consisted of an E.coli strain carrying a growth dependent promoter, in this case the fis promoter, fused to a gene expressing an
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Rapid production of antigen-specific monoclonal antibodies from a variety of animals

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

tissue were suspended in 1 ml PBS-BSA and stained with fluorescently labeled antigen (0.1 µg/ml) and fluorescently labeled antibody against IgG at 4°C for 30 minutes with gentle agitation. After washing with PBS, the cells were stained with ER-tracker as described above. The forward- versus-side-scatter (FSC vs SSC) lymphocyte gate (R1) was applied to exclude dead cells. The PCs (IgG low ER high , R2 gate) were further subdivided into fractions according to their binding levels of fluorescently labeled antigens to define the ASPCs (IgG low ER high antigen + ) and non-speci- fic PCs (IgG low ER high antigen - ). Single-cell sorting was performed using a JSAN flow cytometer equipped with an automatic cell deposition unit (http://baybio.co.jp/english/ top.html) with fluorescently labeled antibodies against IgG monitored in the FL-l (mouse, human and guinea pig) or FL-5 (rabbit and rat) channel, fluorescently labeled antigen in the FL-1 (GFP Dylight 488) or FL-2 (insulin-Cy3) chan- nel and ER-tracker in the FL-7 channel.
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Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

incubating the cells for 40 min at 37 °C. Thereafter, stained cells were centrifuged (at 1500 rpm for 5 min) and pellet was suspended in 500 μl of PBS for flow analysis. The DNA content was then quantified using a FACScaliburTM flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with argon laser (488 nm). While running the cytometer, dot plots were displayed to identify the cells based on their physical parameters on the forward scatter light (FSC) vs. side scatter light (SSC). To evaluate cell cycle progression, samples were acquired with the FL-2 fluorescence channel set to a linear scale, which allows better characterization of the G0/G1 and G2/M DNA peaks. Pulse processing was used to exclude cell doublets and clumps from the analysis. This can be achieved by using pulse area vs. pulse width. For the analysis, a gate was set on the single cell population using pulse width vs. pulse area. Then, this gate was applied to the scatter plot to gate out obvious debris. Thereafter, gates were combined and applied to the PI histogram plot. To quantitate the percentage of cells in each cell cycle phase, markers were set within the analysis program.
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Using Fluorescent Microspheres as a Non-Biological Surrogate Indicator for Sequential Disinfection Performance.

Using Fluorescent Microspheres as a Non-Biological Surrogate Indicator for Sequential Disinfection Performance.

The fluorescence average intensity decay with an ozone concentration of 0.26 and 0.66 mg/L for ozone pretreated microspheres at pH 7 and 20 ± 2ºC is illustrated in Figure 3A. The convergence between the data depicted in Figure 3A confirms that the Ct concept is applicable to microspheres exposed to ozone. Since the pretreatment stage was easier to control at the lower ozone concentration, the 0.30 ± 0.03 mg/L was selected for all subsequent pretreatments. The ozone pretreatment time was determined by trial and error. A pretreatment time between 90 to 100 minutes appeared to work best for representing Cryptosporidium inactivation. However, the selected pretreatment time was lower than what was reported in the literature [14]. In this study, the results suggest that the microsphere inactivation has the same decay rate independent of the initial fluorescence intensity for pretreatment times greater than 90 minutes. However, a different flow cytometer and software was used in this study than what was reported in the literature. Different flow cytometers have different relative fluorescence scales/FITC resolution available. Fluorescence intensity values are directly related to the instrument settings, and for that reason, threshold values are not absolute and differ depending on the flow cytometer settings.
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Use of Formalin-Fixed, Propidium Iodide-Stained Human Leukocytes as a Standard for Enumerating CD4+ T Lymphocytes in a Single-Platform Assay

Use of Formalin-Fixed, Propidium Iodide-Stained Human Leukocytes as a Standard for Enumerating CD4+ T Lymphocytes in a Single-Platform Assay

The single-platform method does not involve a hematology analyzer, using only a flow cytometer. It is more precise as it relies on either concomitant precise measurement of the fluid volume in which such suspended cells are enumerated or the precise addition of fluorescent calibration particles to the sam- ple. While a new generation of flow cytometers with precision fluidics may represent the long-term solution to problems of interlaboratory variation, most laboratories still rely on flow cytometers that lack the ability to measure fluid volumes pre- cisely.

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Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up

The single-cell fluorescence intensities were measured in engineered E.coli cells where the signal can be transcrip- tionally induced via exogenous IPTG (Fig. 2). This choice is particularly convenient since a modification of the inducer concentration produces a change in the stat- istical moments of the fluorescence distribution, allow- ing the set-up validation over a wide range of signal’s intensities using a single gene circuit, thus avoiding biases introduced by different topologies or environmen- tal conditions. Data are expressed as average value ± standard error (SE). The squared coefficient of variation (CV 2 ) was used to quantify biological noise, since it is a measure of the signal’s dispersion around its average value. To assess the capability of the proposed method to provide a reliable, low-cost alternative to a flow cyt- ometer, we validated the set-up described above compar- ing its results with those obtained with a flow cytometer, the gold standard for the acquisition of single-cell fluor- escence. Both datasets were normalized with respect to the average fluorescence intensity of the tested circuit at the highest level of induction. The same number of cells (~12 x 10 3 ) was used for the acquisition of each induced fluorescence level with the microscopy set-up. This facili- tates the comparison among different experimental condi- tions while preventing distortions introduced by the different cardinality of the tested populations. This number of cells was the minimum value required to obtain a stable relation with flow cytometry measurements (Fig 5a).
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ANTIPROLIFERATIVE AND APOPTOGENIC EFFICACY OF ANTIDIABETIC DRUGS METFORMIN AND SITAGLIPTIN AGAINST MCF7 AND HEPG2 CANCER CELLS: A COMPARATIVE MOLECULAR STUDY

ANTIPROLIFERATIVE AND APOPTOGENIC EFFICACY OF ANTIDIABETIC DRUGS METFORMIN AND SITAGLIPTIN AGAINST MCF7 AND HEPG2 CANCER CELLS: A COMPARATIVE MOLECULAR STUDY

AnnexinV-FITC/PI binding assay by flow cytometer demonstrated that only metformin but not sitagliptin could induce early apoptotic cell death in breast cancer cells but it could vastly induce the late apoptotic signs in MCF7 cells whereas in liver cancer cell lines sitagliptin, like metformin and pioglitazone, was observed to induce cell death, both early and late apoptosis, significantly. Metformin and Sitagliptin induced Capase3 activity dose dependently though sitagliptin did show a significantly lower effect than the other drugs. But to summarize the experiments, it should be emphasized that sitagliptin, unlike previous reports from various investigators 2, 18-22 , has been observed to show cell-death inducing characters.
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High-resolution underway measurements of phytoplankton photosynthesis and abundance as an innovative addition to water quality monitoring programs

High-resolution underway measurements of phytoplankton photosynthesis and abundance as an innovative addition to water quality monitoring programs

Biomass is an important parameter to understand the role of phytoplankton in the ecosystem and biogeochemical cy- cles. Its direct measurement using high-resolution methods is challenging. Chlorophyll a concentration is often used as an estimate for biomass, although the carbon : Chl a ra- tio is dependent on abiotic conditions and species-specific phenotypic plasticity, and chlorophyll a is therefore not di- rectly related to biomass (Flynn, 1991, 2005; Geider et al., 1997; Alvarez-Fernandez and Riegman, 2014; Halsey and Jones, 2015). In this study, chlorophyll concentrations are estimated by red fluorescence, which results in a good fit for both the FRRf (adjusted R 2 = 0.66) and the FCM (ad- justed R 2 = 0.90). The impact of abiotic conditions on fluo- rescence as a predictor for chlorophyll a content was tested by comparing the relationship in the different months. Only the flow cytometer data were significantly affected by en- vironmental conditions. The different environmental condi- tions per month did not affect the regression line of the FRRf data. Since the two instruments differ in optics as well as measurement setup (measurements per cell vs. bulk), differ- ences are not surprising. The different measurement setup, with the flow cytometer measuring the fluorescence per par- ticle, while the FRRf does a measurement of the bulk sam- ple, might blur the effect of environmental conditions. In a bulk measurement, other particles in solution scatter the excitation and emission photons, plus the emitted fluores- cence of the phytoplankton is subject to reabsorption, espe- cially at higher biomass densities. Yet, the difference most affected by environmental conditions is the fluorescent state of the photosystems. The strong laser of the flow cytometer can only measure the maximum fluorescence (F m ), which
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Variance in multiplex suspension array assays: A distribution generation machine for multiplex counts

Variance in multiplex suspension array assays: A distribution generation machine for multiplex counts

This M&M metaphor is analogous to how microspheres are presented to the flow cell of a Luminex flow cytometer: For the Luminex system, a microsphere assay mix is made with the intent that equal numbers of each bead classifier be present in the mix. In the above metaphor, we have the same number of each M&M color in the mix. In the Luminex system, classification of microspheres in the mix is by the ratio of intensity of 2 fluorophores bound into in the surface of the microspheres. In the above metaphor, classification is by color of each M&M candy. In the Luminex system, a sample of a microsphere mix is pipet- ted into wells in a multi-well plate containing sample. In the M&M metaphor, the well is represented by the swim- ming pool filled with a mix of M&Ms. In the Luminex sys- tem, the flow cytometer's acquisition probe is dipped into a well, sucking up a quantity of sample. In the M&M met- aphor, the large barrel scooping up a sample of the M&M mixture out of the swimming pool corresponds to the probe sucking up sample. Some proportion of the Luminex instrument's acquired multiplexed microspheres from the sample makes it to the flow cell and then are counted after gating. In the M&M metaphor, this corre- sponds to the number of M&Ms that land inside the 6 foot diameter circle.
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Stress Response inCandida albicansInduced byBoric Acid

Stress Response in Candida albicans Induced by Boric Acid

more surviving cell bodies (flow cytometer events) than in water. Water without boric acid showed higher counts and little loss of viability. Programmed cell death may occur through apoptosis or in some cases autophagy, with the latter dependent on nutrient deprivation [22] which may help explain differences between boric acid effects with and without nutrient. Among the responses to stress may be the up- regulation of protective substances such as enzymes that inactivate reactive oxygen species. However, when cells enter the apoptotic pathway, protective substances may be decreased and reactive oxygen species accumulate. A facile method to explore this in boric acid-treated Candida was measurement of catalase activity which was decreased with boric acid treated cells (Fig. 7). This, along with annexin V data supports the possibility that loss of viability in these experiments involved programmed cell death, though additional methods may be used to further explore and confirm the mechanism.
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