Rapid cell count and intactness assessment of pre- and post-Bead Ruptor Elite™ Bead Mill Homogenized neuroprogenitor cells using the Cellometer K2 Fluorescent Cell Counter

Proper cell sample preparation is critical at the front end of any cell-based assays. Specifically, an appropriate cellular lysis procedure is necessary for downstream analyses of intracellular analytes such as DNA/RNA, intracellular proteins, and small molecules (1). However, traditional cellular lysis methods can be time-consuming and heavily reliant on reagents like enzymes or detergents to facilitate lysis. The Omni Bead Ruptor Elite bead mill homogenizer provides a quick and efficient solution for cell lysis that is not dependent on chemical reagents. Cell suspensions can be homogenized in phosphate buffered saline (PBS) on the bead mill in less than 30 seconds, releasing all intracellular analytes of interest into solution and producing homogenate for downstream analysis. 

Scientists require accurate cell count and viability results to prepare cells for downstream assays. Cell viability can be measured by assessing the cell membrane integrity with different commercially available viability dyes such as trypan blue (TB), propidium iodide (PI), or a combination of acridine orange (AO) and PI. Nucleic acid staining dyes like AO/PI have been widely used and proven to be a reliable dual-stain for cell counting and viability measurement. AO is an acidic and hydrophobic molecule that readily passes through cellular membranes, binds to double-stranded nucleic acid, and emits a wavelength of 525 nm (green) when excited with a blue light. Contrastingly, PI is impermeable to viable cells with intact membranes and only passes through compromised cellular membranes, where it binds to the nucleic acid and emits a wavelength of 593 nm (red/orange) when excited with a green light (2,3).

Therefore, nucleated cells with intact membranes stained with AO will fluoresce green. In contrast, membrane-compromised cells will only fluoresce red due to Förster resonance energy transfer (FRET) that facilities the absorption of AO fluorescent signals by PI with minimum spill over. 

In the past decade, the Cellometer® K2 fluorescent cell counter has been employed for rapid cell count and viability measurement and easily distinguishing nucleated cells from cellular debris and micelles using AO/PI (4,5). This is highly important to reinforce the reliability of upstream cell counting results and membrane intactness assessment, as well as to provide scientists with live cell counts to ensure an appropriate amount of cells is utilized depending on the requirements of downstream assays. 

In this work, we demonstrate the use of the Cellometer K2 to determine cell count and viability via AO/PI staining pre-homogenization (intact cells) and post-homogenization (lysed cells) of neuroprogenitor cells produced by the Omni Bead Ruptor Elite bead mill homogenizer. Furthermore, the acquired brightfield and fluorescent images can be used to verify the homogenization and intactness of the cells directly. The cell counter can quickly measure nucleated viable and nonviable cell count results and disregard the non-countable events (debris) during sample preparation that are crucial for the DNA/RNA, intracellular proteins, and small molecule-based downstream assays.

Figure 3: Brightfield and fluorescent images of neuroprogenitor cell sample 1 stained with AO/PI and counted on the Cellometer K2 pre- and post-homogenization on the Omni Bead Ruptor Elite bead mill homogenizer.