Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa

2011 ◽  
Vol 36 (9-11) ◽  
pp. 424-429 ◽  
Author(s):  
Xi Xiao ◽  
Zhi-ying Han ◽  
Ying-xu Chen ◽  
Xin-qiang Liang ◽  
Hua Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Microcystis Aeruginosa ◽  
Metabolic Activity ◽  
Pi Method

scholarly journals Effect of Zinc on Microcystis aeruginosa UTEX LB 2385 and Its Toxin Production

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 92 ◽  
Author(s):  
Jose L. Perez ◽  
Tinchun Chu
Keyword(s):  
Flow Cytometry ◽  
Stress Response ◽  
Microcystis Aeruginosa ◽  
Stress Responses ◽  
Algal Blooms ◽  
Phase Contrast ◽  
Phase Contrast Microscopy ◽  
Zinc Stress ◽  
Cell Quota ◽  
Contrast Microscopy

Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium, Microcystis aeruginosa UTEX LB 2385, by monitoring cells with (0, 0.1, 0.25, and 0.5 mg/L) ZnCl2 treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC50) was 0.25 mg/L ZnCl2. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl2. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl2 treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl2 treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl2 treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl2 control cells and 0.1 mg/L ZnCl2 treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl2 concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl2 treatment were observed.

scholarly journals Evaluation of Alternative Methods to Assess the Biological Properties of Propolis on Metabolic Activity and Biofilm Formation in Streptococcus mutans

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Jorge Jesús Veloz ◽  
Marysol Alvear ◽  
Luis A. Salazar
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Streptococcus Mutans ◽  
Metabolic Activity ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Biological Activities ◽  
Biological Properties ◽  
Alternative Methods ◽  
Flow Cytometry Analysis

Several biological activities have been reported for the Chilean propolis, among their antimicrobial and antibiofilm properties, due to its high polyphenol content. In this study, we evaluate alternative methods to assess the effect of Chilean propolis on biofilm formation and metabolic activity of Streptococcus mutans (S. mutans), a major cariogenic agent in oral cavity. Biofilm formation was studied by using crystal violet and by confocal microscopy. The metabolic activity of biofilm was evaluated by MTT and by flow cytometry analysis. The results show that propolis reduces biofilm formation and biofilm metabolic activity in S. mutans. When the variability of the methods to measure biofilm formation was compared, the coefficient of variation (CV) fluctuated between 12.8 and 23.1% when using crystal violet methodology. On the other hand, the CV ranged between 2.2 and 3.3% with confocal microscopy analysis. The CV for biofilm’s metabolic activity measured by MTT methodology ranged between 5.0 and 11.6%, in comparison with 1.9 to 3.2% when flow cytometry analysis was used. Besides, it is possible to conclude that the methods based on colored compounds presented lower precision to study the effect of propolis on biofilm properties. Therefore, we recommend the use of flow cytometry and confocal microscopy in S. mutans biofilm analysis.

scholarly journals Suppression of Ovarian Cancer Cell Growth by AT-MSC Microvesicles

2020 ◽  
Vol 21 (23) ◽  
pp. 9143
Author(s):  
Agnieszka Szyposzynska ◽  
Aleksandra Bielawska-Pohl ◽  
Agnieszka Krawczenko ◽  
Olga Doszyn ◽  
Maria Paprocka ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Metabolic Activity ◽  
Ovarian Cancer Cell ◽  
Ovarian Cancer Cells ◽  
Target Cells ◽  
Effective Transfer

Transport of bioactive cargo of microvesicles (MVs) into target cells can affect their fate and behavior and change their microenvironment. We assessed the effect of MVs derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) on the biological activity of the ovarian cancer cell lines ES-2 (clear cell carcinoma) and OAW-42 (cystadenocarcinoma). The HATMSC2-MVs were characterized using dynamic light scattering (DLS), transmission electron microscopy, and flow cytometry. The anti-tumor properties of HATMSC2-MVs were assessed using MTT for metabolic activity and flow cytometry for cell survival, cell cycle progression, and phenotype. The secretion profile of ovarian cancer cells was evaluated with a protein antibody array. Both cell lines internalized HATMSC2-MVs, which was associated with a decreased metabolic activity of cancer cells. HATMSC2-MVs exerted a pro-apoptotic and/or necrotic effect on ES-2 and OAW-42 cells and increased the expression of anti-tumor factors in both cell lines compared to control. In conclusion, we confirmed an effective transfer of HATMSC2-MVs into ovarian cancer cells that resulted in the inhibition of cell proliferation via different pathways, apoptosis and/or necrosis, which, with high likelihood, is related to the presence of different anti-tumor factors secreted by the ES-2 and OAW-42 cells.

Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dualgfp-luxAB Marker System

1999 ◽  
Vol 65 (2) ◽  
pp. 813-821 ◽  
Author(s):  
Annika Unge ◽  
Riccardo Tombolini ◽  
Lars Mølbak ◽  
Janet K. Jansson
Keyword(s):  
Flow Cytometry ◽  
Metabolic Activity ◽  
Fluorescent Protein ◽  
Luciferase Activity ◽  
Energy Requirement ◽  
Cell Number ◽  
Energy Status ◽  
Bacterial Strains ◽  
Marker System ◽  
Gfp Fluorescence

ABSTRACT A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxABand gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescensSBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.

Risk Statification of Follicular Lymphomas Using Two Independent Prognostic Factors: Lymphoma Cell Size Assessment by Flow Cytometry and FDG PET Based Metabolic Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5098-5098
Author(s):  
Mohammad Muhsin Chisti ◽  
Govinda R. Brahmanday ◽  
Alaa A Muslimani ◽  
Siddhartha Yadav ◽  
Paul Rigo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
Cell Size ◽  
Metabolic Activity ◽  
Fdg Pet ◽  
Low Risk ◽  
Lymphoma Cell ◽  
Pet Scan ◽  
Low Grade ◽  
Grade 3

Abstract Abstract 5098 Introduction: Follicular Lymphoma (FL) comprises of 20–30% of Non-Hodgkin's Lymphomas. It is the most common lymphoma with varied clinical aggressiveness, depending on morphological grading on histology. The prognosis of FL is heterogeneous. Therefore therapeutic options for FL are increasingly becoming more risk-adopted. Pathologically, FL has been graded into grade 1–2 (FL1–2) and grade 3 (FL3) by visual counting or estimating the absolute number of centroblasts in neoplastic follicles. Recent studies found that the average tumor cell size of FL is highly variable, but is consistently reproducible, achieved by an objective measurement using flow cytometry. A significant variation in metabolic activity has also been observed in FL studied by PET scan. The purpose of this study was to investigate a new risk stratification method for FL using a combined criterion of Flow cytometry based cell size of FL cells and metabolic activity based on FDG PET scan to further evaluate their prognostic values. Materials and Methods: Retrospective study of 64 patients (25 males and 39 females with median age of 61 years), diagnosed with FL between 2006 –2010 in Beaumont Health System with proper HIC approval was performed. 44 cases were graded morphologically into low risk (FL 1–2) and 20 into high risk (FL 3), based on current WHO classification. The average tumor cell size was measured based flow cytometric analysis of the relative forward scatters (FSC) of tumor cells relative to that of internal T-cells in the lymphoma tissue. The metabolic activity was measured as in maximal standardized uptake value (SUV max) of the involved lymph node by PET scan based F-18 FDG uptake. The SUV was normalized to a glucose level of 100 using the formula: SUV100={[100 mg/dl]/[glc]}g where g=[-0. 5] from our prior publication and [Glc] was the glucose level of the patient recorded at the time of PET scan. The difference of average lymphoma cell size between grade 1–2 (FL 1–2, indolent) and grade 3 (FL 3, aggressive) was compared with Student t test. The difference of size distribution between FL with low SUV100 and FL with high SUV100 were compared with Chi square and Kappa tests. A two-tail p-value less than 0. 05 was considered significant in all tests. FSC and Metabolic activity were cross tabbed and correlated using cut off values determined by discriminant analysis. Results: The FSC of B-cells were significantly lower in low grade FL than that in high grade FL (p=0. 00002). In contrast, FSC of residual T-cells in low grade FL and high grade FL were about the same (p=0. 2). Average lymphoma cell size of the low risk group (relative FSC of −15, range of −83 to 153) was significantly smaller than that of high risk group(relative FSC of 92, range of −32 to 243), (P<0. 0005)(figure 1). Using a cut off value FSC= −18 from discriminant analysis, and group them into aggressive and indolent FL, there was significant difference in metabolic activity (SUV 100) between the lymphoma with small and large tumor cell sizes (p=0. 021, by Chi-Square test)(figure 2). Combining these two objective independent measurements could accurately place low risk FL into observation with 95% confidence. Conclusion: The significance of average lymphoma cell size identified by FSC in this study between FL grade 3 and grade 1–2 validate potential utility of flow cytometry in grading FL in refining the grading because the measurement of average lymphoma cell size by flow cytometry it is more reproducible, objective, and easy to use than manual counting centroblasts microscopically. Since increased metabolic activity is often associated with more aggressive clinical behavior, combining tumor cell size by flow cytometry and metabolic activity by FDG PET imaging can potentially serve as an independent criterion in risk stratification and better grading of FL. Further clinical correlation is in progress. Disclosures: No relevant conflicts of interest to declare.

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