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

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.

MATERIALS AND METHODS

Comparing cell counting accuracy of the LUNA™ Reusable Slide and a hemocytometer

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.

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 bluewere 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.

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.

Figure 1. Comparison of the cell count results of the LUNA™ Reusable Slide and a hemocytometer.

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.

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. Compatibility 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™)

REFERENCES
Tucker KG, Chalder S, Al-Rubeai M, Thomas CR. Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis. Enzyme Microb Technol. 16:29–35. (1994)