Use of Fluorescent Probes for Determination of Yeast Cell Viability by Gravitational Field-Flow Fractionation

Abstract

The quality of wine greatly depends on the features of the yeast used in its production, and yeast cell viability is one of the most important quality control issues to consider in this regard. In the first steps of winemaking, the use of a low-cost and simple methodology for monitoring the cell viability of yeast inoculates is of paramount importance. Gravitational field-flow fractionation is a useful technique for the determination of cell viability because it provides gentle experimental conditions, although the proper use of fluorophore probes as biomass indicators is required. In this paper the use of different fluorescent probes such as carboxyfluorescein diacetate (cFDA), calcein-AM, and SYTO-13 were considered as viability biomarkers. Calceina-AM allowed the establishment of a direct GrFFF method to determine cell viability, with a limit of detection of 5.0 x 10(4) viable cell/mL. SYTO-13 could be used as biomass indicator with a limit of detection of 3.5 x 10(4) total cells/mL. The suitability of the procedure was tested with three commercial yeast samples, and the results were compared with those obtained using standard techniques.

Exometabolome analysis reveals hypoxia at the up-scaling of a Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process

“…The S. cerevisiae cell viability was measured using a BD FACS Calibur Flow Cytometer (BD Bioscience, San Jose, CA, US) as described in Garcia et al ., [35] and the manufacturer’s instruction. Samples taken from the culture were immediately diluted with Phosphate Buffered Saline (PBS; pH 7.2) to a final concentration of ~10 6 cells mL −1 and stained with the red fluorescence dye, propidium iodide (PI).…”

Analysis of relationship between cell cycle stage and apoptosis induction in K562 cells by sedimentation field-flow fractionation

“…These advantages are based on the drastic limitation of cell-solid phase interaction by the use of: (1) a specific separation device: empty ribbon-like channel without stationary phase and (2) a device setup allowing the "Hyperlayer" elution mode, a size/density driven separation mechanism. Since the report of Caldwell et al [14] on mammalian cells, FFF, SdFFF and related technologies have been used in many biological fields such as hematology, microorganism analysis, biochemistry/biotechnology and molecular biology, neurology and cancer research [4,20,22,[31][32][33][34][35][36][37][38][39][40][41][42][43]7,[44][45][46][47][48]. Over several years, we have studied the use of SdFFF in cancer research to study chemical induced apoptosis or differentiation in cancer cell lines.…”

Monitoring of barley starch amylolysis by gravitational field flow fractionation and MALDI‐TOF MS

“…Extremely low cost and suitability for separation of micrometer‐sized particles makes this method attractive for characterization and separation of a wide range of inorganic or organic analytes. Gentle experimental conditions (low pressure and weak force field) and the possibility of using isotonic buffer solutions as carrier liquids make GFFF unique and suitable also for separation and purification of biological samples such as living blood or stem cells,15, 16 parasites,17 yeasts18–20 and starch granules 9, 21–25. Sedimentation FFF was used for monitoring of starch amylolysis of model starch granules obtained from rice26, 27 and wheat 28…”