**:Diancheng Biotechnology Diancheng Case |Yeast cell counts are performed using an automated cell counter
Original link:Welcome to pay attention to Hongke and provide you with the latest information!
Yeast can be used in a variety of applications, including basic research, brewing and distillation, and food production, all of which require accurate cell counting and viability determination. The smaller size and morphology have proven to be quite challenging for automated cell counters, and methods for manually counting yeast utilizing live stains are cumbersome and error-prone. In this post, we share how to accurately and reliably count yeast cells with the LUNA FX7 Automated Cell Counter.
1 Inoculate the yeast strain EC1118 into YPD broth and culture at RPM overnight.
2. Dilute the yeast in 1xPBS in a ratio of 1:3 to 1:5.
3 The diluted culture is diluted into 2 equal samples, and then one sample is heat-inactivated (65, 20 min) to generate "live and dead cells" for viability testing.
4 Use yeast cell buffer to further dilute the cells in the ratio of cells:buffer = 1:5 and 1:10.
5 Viability test: Mix live and dead cells as shown in the table.
1 Linearity of cell viability measured by the LUNA-FX7 Automated Cell Counter.
To determine the linearity of cell viability, samples of various viabilities were prepared using live and dead cells and then counted using the Luna-FX7 automated cell counter. With LUNA-FX7, viability can be checked in fluorescence mode or [Cell counting & viability] in brightfield mode.
To determine cell viability in brightfield mode, mix the yeast sample with the same volume of 0Mix 02% methylene blue stain and load 10 L of sample onto a LUNA cell counting slide. Yeast samples were prepared as dead cells, % live cells (100%).
Use the LUNA-FX7 automated cell counter.
Cell counting & viability in brightfield mode was used to determine cell viability.
Yeast cells stained and labeled with methylene blue.
As shown, 5 samples were replaced with 002% methylene blue stained and imaged in brightfield mode [cell counting & viability]. Count 3 times to calculate the average viability and confirm the linearity of cell viability accordingly. Vitality R2 = 09964, which is highly consistent with the theoretical vitality.
Use the LUNA-FX7 automated cell counter.
Cell counting & viability in brightfield mode was used to determine cell viability.
The linear plot confirms that the measured viability is versus the theoretically expected viability (r2=0.).9964)
Immediately after, to study cell viability in fluorescence mode, 5 yeast samples were stained with AOPI stain and 10 L samples were loaded into PhotoSlide slides. After AOPI staining, images are taken and observed.
As shown in the figure below, 5 samples with different viabilities were counted 3 times, and the measured yeast cell viability was R2=09979, which has a significant correlation with theoretical vitality.
Use the LUNA-FX7 automated cell counter.
Cell viability is determined by [Cell Lines & Primary Cells, Advanced] in fluorescence mode.
Yeast cells stained and labeled with acridine orange propidium iodide.
Use the LUNA-FX7 automated cell counter.
Cell viability is determined by [Cell Lines & Primary Cells, Advanced] in fluorescence mode.
The linear plot confirms the measured viability and the theoretically expected viability (r2=0.).9979)
2 Linearity of cell concentration analysis on the LUNA-FX7 automated cell counter.
To determine the linearity of different concentrations when counting yeast using the LUNA-FX7 automated cell counter, high concentrations of yeast were prepared and concentrations were measured by serial dilutions.
For observation in brightfield mode, use 002% methylene blue stained and counted in [total cell counting] and [cell counting & viability] in brightfield mode, respectively.
As you can see from the graph, the measured R2 values are 0998 and 09991。This is also observed in fluorescence mode, where the R2 value is 09753。This means that yeast counts with LUNA-FX7 show high linearity from high to low concentrations, and more accurate determinations can be made with LUNA-FX7.
Through these experiments, we can think about the limits of yeast cell quantification. Yeast cells have an average cell size of 3 4 m, and a typical mammalian cell size is 10 m. Thus, with 2-channel slides in mammalian cells500e+4 to 1Compared to the range of 50 e+7 cells ml, yeast cells can be adjusted up to 500e+5 to 300e+7 cells/ml。
Measure the concentration of yeast samples in brightfield mode on the LUNA-FX7 Automated Cell Counter.
Total cell count.
Cell count and viability.
Measure the concentration of yeast samples in [Cell Lines & Primary Cells, Advanced] in fluorescence mode on the LUNA-FX7 automated cell counter.
3 Comparison of the LUNA-FX7 automated cell counter with a hemocytometer.
We performed an experiment to compare the conventionally used hemocytometer with the LUNA-FX7.
As in the previous experiment, the sample was sampled with 0Stain with 02% methylene blue and AOPI and load 10 L each onto hemocytometers, Luna, and Photonslide slides. Images were taken in both brightfield and fluorescence modes of LUNA-FX7 and the concentrations were compared to those measured on a hemocytometer. As shown, no significant difference was observed between the values measured by the hemocytometer and those measured by the LUNA-FX7.
Comparison of yeast counting results from a hemocytometer and a Luna-FX automated cell counter.
Conclusion. In this experiment, we demonstrated that LUNA-FX7 can accurately and reliably count yeast cells. In addition, yeast counting with LUNA-FX7 is 20 times faster, more reliable, and less error-prone than traditional manual counting methods.