APPLICATION  

Using the LUNA™ Reusable Slide for Accurate Cell Counting with Automated Cell Counters

#Bioprocess monitoring #biomanufacturing #cell therapies #bioactive production #automatic calculation #elapsed time #growth rate #doubling time #growth curves

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INTRODUCTION  
Cell counting is an essential step in routine cell maintenance and for obtaining accurate and consistent experimental results. In recent years, automated cell counters have become more popular because affordable instruments have become increasingly available at competitive prices. However, many researchers still use the hemocytometer to avoid the expenses associated with the use of disposable slides. To overcome this problem, Logos Biosystems designed a reusable slide (LUNA™ Reusable Slide, L12008) to be used with the LUNA™ family of automated cell counters. Here, we compare the cell counting results of the LUNA™ Reusable Slide, a hemocytometer, and disposable slides.  
RESULTS Cell counting accuracy of the LUNA™ Reusable Slide   
To determine the cell counting accuracy of the LUNA™ Reusable Slide, the total cell concentration results obtained with the LUNA™ Reusable Slide were compared to those obtained manually with the hemocytometer. The LUNA™ Reusable Slide results were highly correlated (R2 > 0.999) with the hemocytometer results at the tested concentrations (Fig. 1). The cell concentration data for the LUNA™ Reusable Slide were more consistent at higher concentrations (>2 x 106 cells/mL) than the hemocytometer. The fact that the LUNA-II™ counts a larger area within the LUNA™ Reusable Slide than was manually counted with the hemocytometer may account for the difference.
Cell counting performance of the LUNA™ Reusable Slide for brightfield counting
To assess the compatibility of the LUNA™ Reusable Slide for automated brightfield counting, cells stained with trypan blue were loaded into the LUNA™ Reusable Slide and disposable slides and counted with the LUNA™, LUNA-II™. As shown in Fig. 2, there was no significant difference in the total, live, or dead cell concentrations among the slides or devices.
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MATERIALS AND METHODS Comparing cell counting accuracy of the LUNA™ Reusable Slide and a hemocytometer   
To determine the accuracy of cell counts done with the LUNA™ Reusable Slide, cell counts were compared to those obtained manually with a hemocytometer. Cells were serially diluted and mixed with an equal volume of 0.4% trypan blue stain. 10 µL of the mixture was loaded into the LUNA™ Reusable Slide and a glass hemocytometer with Neubauer counting grids (Marienfeld). To obtain cell concentrations with the glass hemocytometer, the hemocytometer was imaged with the iRiS™ Digital Cell Imaging System and the cells within the four corner squares of the Neubauer counting grids were counted. Cell samples in the LUNA™ Reusable Slide were counted with the LUNA-II™ Automated Cell Counter. All experiments were performed in triplicate.
Evaluating cell counting accuracy of the LUNA™ Reusable Slide with brightfield and fluorescence automated cell counters
The performance of the LUNA™ Reusable Slide for brightfield and fluorescence cell counting was evaluated by using two stains, trypan blue and acridine orange/propidium iodide (AO/PI), respectively. For brightfield counting, cells mixed 1:1 with trypan blue were loaded into the LUNA™ Reusable Slide or disposable slides and counted with the LUNA™, LUNA-FL™, and LUNA-II™ automated cell counters. For fluorescence cell counting, 2 µL AO/PI was mixed with 18 µL cell suspension and 10 µL was loaded into the LUNA™ Reusable Slide or disposable slides and counted with the LUNA-FL™ Automated Fluorescence Cell Counter. All experiments were performed in triplicate.  
  • Figure 1. Comparison of the cell count results of the LUNA™ Reusable Slide and a hemocytometer.
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  • Figure 2. Compatibility of the LUNA™ Reusable Slide with automated cell counters by trypan blue staining. (Top) Cell images captured and tagged by the LUNA™ family of automated cell counters; (bottom) cell counting results using the LUNA™ Reusable Slide and disposable slides.
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Cell counting performance of the LUNA™ Reusable Slide for fluorescence staining
To assess the compatibility of the LUNA™ Reusable Slide for automated fluorescence counting, cells stained with AO/PI were counted with the LUNA-FL™. There were no significant differences in any of the parameters (Fig. 3).
  • Figure 3. CCompatibility of the LUNA™ Reusable Slide with automated cell counters by AO/PI staining. (Top) Cell images captured by the LUNA-FL™; (bottom) cell counting results using the LUNA™ Reusable Slide and disposable slides.
CONCLUSIONS  
Automated cell counting with the LUNA™ Reusable Slide is a good alternative to the hemocytometer for the following reasons:
No learning curve – similar to using a hemocytometer
Reusable and cost-effective
Accurate cell counting results
Removal of user variability and time consuming calculations
Compatible with autofocused automated counting (with the LUNA-II™ and LUNA-II YF™)