Application Note Yeast Analysis with a life - dead staining kit (Yeast control - viability) (CyFlow Cube 8 and CyFlow Cube 6 with CyView TM version 1.5 CyFlow Space) Introduction The Partec Yeast control - Viability staining kit aims to determine the percentage of living yeast cells from a growing culture or from transport competent yeast cells intended to be used for cultivation at a later stage (income quality control of yeast cultures). The result of such a measurement is the total amount of yeast cell (cell concentration) and the ratio of living cells out of such a culture. Materials, Instrument and Methods Yeast cells: as a standard control please use fresh baker s yeast from the supermarket. Please do not use the dry yeast but the fresh, dehydrated package (maximum usage after purchase: for 10 days). Yeast control viability staining kit (product code 05 6000 02). The staining kit consists of 2 reagents: reagent A and reagent B. Additional material required but not provided by the staining kit: PBS The sample should be analyzed on a flow cytometer with a blue laser excitation light source (488 nm wavelength) capable of detecting FCS, SSC as well as green (FL1, interference bandpass filter 536 / 40 nm) and orange / red fluorescence (FL2, interference bandpass filter 590 / 50 nm or FL3, interference bandpass filter 675/20 depending on instrument type). The following examples will highlight measurements on a CyFlow Cube and a CyFlow space instrument. Reagent Specification Spectral characteristics Reagent A Live cell stain Comparable to Fluorescein isothiocyanate (FITC) Absorption max: 495nm Emission max: 519nm Reagent B Dead cell stain Table 1: Description of staining reagents Propidium iodide (PI) Absorption max: 530nm Emission max: 630nm 1/6
Sample preparation Cell suspension Starting with commercial dehydrated yeast: resuspend 0,01 g (10 mg) of yeast material in 10 ml PBS Starting with a yeast cell culture: take 10 µl of cells from a growing culture in mid-log phase and dilute with 990 µl PBS (dilution factor: 100) Staining reagents Take 1 ml of yeast suspension (either from the commercial dehydrated yeast or the growing yeast culture) and add 10 µl of reagent A. Incubate for 5 min at room temperature protected from light. Add 10 µl of reagent B and incubate for another 5 min at room temperature protected from light. The solution B contains propidium iodide and methanol and should be handled with care. Please follow the instructions given within the product leaflet. The sample is ready for analysis. Prepared samples should be analyzed within 15 min after preparation. Optional sample preparation: as a dead cell control prepare one lot of cells as described above and incubate the cells in an appropriate, heat-proof sample container at 70 C in a water bath for 10 min. In such way obtained cells can be mixed with fresh cells to generate a control sample consisting of living and dead cells. Analysis on a FCM Set-up of the instrument: 1. Select a layout with three or more plots (histogram and dotplots) showing at least FSC SSC and FL1 FL2 or FL3 (depends on the filter set-up) 2. Select FSC as trigger parameter LL = 100 log3 on FSC and SSC gain approximately 200 log4 on fluorescence parameters gain approximately 330-380 Set the analysis speed to 1 µl / s 3. Attach a negative control sample without stain to fine adjust GAIN values and LL-value Observe the analysis rate (events / sec). If the value exceeds 1000 dilute the sample by a factor of 5 with PBS (and repeat again if the analysis speed is still too high). For later counting analysis keep rate / s below 1000! 2/6
Set the gain values of FSC and SSC in such a way that the cell population shows up in the center of the FSC SSC dotplot (fig. 1). Surround the yeast cells by a polygon and apply this polygon to the dot-plot FL1 vs. FL2/3. Set the gain values of FL detectors to display cells in the first of 4 log decades (fig. 1b) Yeast cells Background a) b) Fig. 1 FSC SSC plot allows identification of the yeast cells (a) which can be surrounded by a polygon and displayed in the fluorescence dot-plot (b) 4. Attach sample stained with FDA only (control), analyse and stop. Set compensation for FL1 vs FL2/3 Fig. 2 FDA vs PI plot (live vs dead) uncompensated without (a) and with compensation line for FDA vs FL3 (b) 5. Attach sample stained with PI only (control), analyse and stop. Set compensation for FL2/3 vs FL1. If required correct GAIN and LL values. 3/6
Fig. 3 FDA vs PI plot (live vs dead) uncompensated with compensation line for PI vs FL1 (a) and compensated (b) Sample analysis and counting: Analyse double stained samples. Use True Volumetric Counting to enumerate number of vital and dead cells (instructions below). Within the dotplot FL1 FL2/3 the fluorescence signals of vital and dead cells can be clearly identified (fig. 4) as vital cells are mainly green fluorescent whereas dead cells are mainly red fluorescent. Fig. 4 FDA vs PI plot (live vs dead) compensated without absolute counting results 4/6
Counting with Electrode Sensors (True Volumetric Absolute Counting, TVAC) In order to quantify the concentration of yeast cells (and vital cells) the measure mode Volumetric counting with electrodes should be used. Fill 850µl of sample in a standard sample tube. Mix well and attach it to the sample port. An analysis cycle according the below sequence is started automatically and a true absolute volumetric counting begins when the liquid level in the tube reaches the first, shorter sensor. 1. The system starts with an initialization sequence of Pre-run phase followed by 2. Stabilization switching into 3. Run phase (a pre-counting phase). When the liquid level in the sample tube reaches the start electrode the counting procedure initiates and the system switches from Run to 4. Count phase leading to the end of counting when the liquid level reached the second sensor 5. During Repositioning sample pump is moving into start position followed by 6. Clean which washes the sample port and flow cell 7. Ready is displayed when the system is ready to analyse the next sample The current phase is indicated always in the Instrument Settings Box next to the flow counter. With sample speed of 3 µl/s this procedure takes about 100 seconds. In order to minimize effects of particle sedimentation during the process it is recommended to use high speed values of 3-5 µl/s! The measured volume is fixed to 200 µl by the position of the electrodes. Start Count Stop 5/6
Cleaning and Shutdown process set speed = 4 µl/s o o Connect a sample tube filled with 1.5ml 0.5 % Hypochloride (Order No. 04-4012) to disinfect the tubing run until system stops Connect a sample tube filled with 1.5ml Cleaning Solution (green solution, Order No. 04-4009) run until system stops o Connect a sample tube filled with 1.5ml Decontamination Solution (violet solution, Order No. 04-4010) run until system stops o Connect a sample tube filled with 1.5ml Sheath fluid (Order No. 04-4007) run 2 minutes and STOP - Shut down FloMax - Switch off CyFlow Space instrument - Shut off the computer 6/6