The detection of reactive oxygen species (ROS) using 2',7'-dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 μM) for 45 min, and then exposed to TBHP (0-50 μM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 μM DCFDA with 25 μM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology.

Ovarian follicle growth and oocyte maturation depend on the viability of granulosa cells (GC). We quantified GC in whole mouse follicles. Single follicles were isolated from adult mouse ovaries and stained with DAPI or Live-Dead stain before fixation. An objective image analysis protocol for counting fluorescent labeled GC was developed that used Image J software to measure GC cytoplasmic and nuclear areas. These data were compared to the number of GC obtained by disaggregating 96 follicles with enzymes to produce a suspension of GC, which then was stained with trypan blue and assessed using a hemocytometer. We found a linear relation between GC/follicle and follicle diameter. Viability of GC/follicle ranged from 40 ± 11 to 72 ± 7%. The coefficient of variation for image analysis of DAPI stained GC by different assessors was 4%, but the number of GC obtained from image analysis was approximately 50% less than from disaggregated follicles. The number of GC in intact mouse follicles was greater than the number reported earlier for fixed ovarian sections. We found that the number of GC was less in fluorescence labeled follicles; it is possible that the three-dimensional structure of the intact follicles obscured the fluorescent signal. Direct quantification of viable GC isolated from follicles appears to be the most accurate method.

Background: Glucose variability is an indicator and independent predictors of mortality and severity of sepsis in critically septic patients of intensive care unit. Objectives: This study evaluated the relationship of glucose variability in critically ill septic patients in relation with serum lactate. Method: It is a prospective observational study was conducted in the Intensive Care Unit (ICU) in Department of Anesthesia, Pain, Palliative Care and Intensive Care, Dhaka Medical College hospital, Dhaka over a period of one year in between 1st January- 31st December, 2015. Total 51 septic adult patients were included in the study according to the selection criteria. In this study, 8 consecutive capillary blood samples were taken with a periodic interval of 3 hours starting from admission. Mean and standard deviation (SD) blood glucose were computed to see the glucose variability and agreement done with serum lactate and severity of sepsis. All collected data were registered documented and analyzed in the statistical program Statistical Package for Social Science (SPSS) version 20.0. Result: Among total 51 patients, 52.9% study cases were of 4th decade with a mean age of 46±12 years had a mild female predominance. Glycemic variability was taken as >2SD. High glycemic variability observed in 70.6% cases. Good agreement observed in glucose variability with serum lactate by Kappa Statistics. Conclusion: Glucose variability shows the prediction of sepsis and it could be used as a useful alternative tool to predict sepsis and severity of sepsis. Bangladesh Crit Care J March 2022; 10 (1): 10-14