Cell Viability Staining
Relevant Instruments:
Click below for the full applications note:
Cell viability assessments are a cornerstone of every cell-based biological endeavor, be it research or biotech companies or advanced therapeutics. Anything that involves the use of cells requires quantifying the cells and determining their status, or measuring the number of alive vs. dead cells. As such, success in these assessments requires solving two primary criteria: accurate cell counts and concentration measurements as well as reliable quantification of viable cells.
In terms of accurate cell counts, ORFLO’s Moxi instruments provide an automated physics-based counting method of thousands of cells based on the Coulter Principle. While many cell counting methods work, the Moxi instruments provide the most accurate estimates of cell concentration on the market.
For viability staining, multiple options exist with different fluorescent and staining characteristics. Both the Moxi V and the Moxi GO II are capable of detecting any viability staining dyes that emit orange/red light that can be detected with the 561nm/LP filter on either instrument. The primary dye that fits these criteria is propidium iodide (PI). The Moxi GO II’s 525/40nm filter will be able to detect any stains that emit green light. These dyes include the most frequently used acridine orange (AO) or calcein AM, however other stains such as MitoTracker™ Green FM or SYBR™ Green will work as well.
Propidium Iodide (PI)
Fig. 1: PI viability measurements on CAR-T cells with a Moxi GO II.
The most commonly used substance for delineating which cells are alive or dead is propidium iodide (PI). ORFLO’s PI-based Moxi Cyte Single Color Viability Reagent (MXA055-3) provides the easiest way to perform single-color viability measurements on either a Moxi V or Moxi GO II. Both instruments can easily detect the fluorescent signal given off from PI-bound dead cells with their 561/nm/LP filter.
This data is from a user performing viability assessments with PI on CAR-T cells with a Moxi GO II (Fig. 1). With active gating capabilities, it is easy to remove the debris (small events on the left of the x-axis) and isolate only the real cells. Further gating on the y-axis (dotted red line) allows for the second layer of analysis with proper delineation of live and dead cell populations. Live cells are PI negative (no fluorescent signal) and dead cells are PI positive. Other instruments use a set threshold fluorescence intensity to measure fluorescence events which makes them prone to miscounting and other errors in the overall viability assessment. However, the Moxi instruments allow the user to fully control gating and other parameters to ensure robust results and achieve specific analysis requirements.
Acridine Orange (AO)
Fig. 2: Viability measurements using ORFLO’s Moxi Cyte Two Color Viability Reagent
While PI identifies dead cells, another marker is often needed to ensure all of the events counted in the lower population of the instrument’s data analysis software are indeed both cells and/or alive. This is where a second marker is required and the complexity of the cell health assessment increases. Acridine orange (AO) is often used as this second marker since it binds to all cells, and is the second component of ORFLO’s Moxi Cyte Two Color Viability Reagent (MXA069-2) along with PI. This dual-color staining kit can only be performed on the Moxi GO II. This data was shared from a user performing an AO/PI experiment on Jurkat cells using the Moxi GO II’s “GO Flow” app and ORFLO’s Moxi Cyte Two Color Viability Reagent. The double positive event population (AO+/PI+, top right quadrant) will be the true dead cells, and the AO+/PI- event population will be all of the true live cells (bottom right quadrant). This example shows a relatively unhealthy cell population with 53.7% dead cells and 46.2% live cells.
Calcein AM + PI
Fig. 3: Viability measurements of cells using calcein + PI
The second green fluorescent option is calcein AM. It is different from AO in that it will only fluoresce in live cells. Like AO, the excitation and emission spectrum is perfect for the Moxi GO II’s 488nm laser and 525/45nm filter and does not overlap with PI. When running a sample on a Moxi GO II with AO/PI, an easy to read two-dimensional output is produced that allows for easy isolation and counting of both true live and true dead cells. This is made even easier by the Moxi GO II’s “Cell Health (Calcein AM Viability)” app, which delineates which cells are which in each quadrant (Fig. 3). This data was shared by a user utilizing their Moxi GO II with their cell culture sample before sending it for FACS sorting. The live cell population is calcein AM+/PI- in the bottom right, and the dead cell population is the calcein AM-/PI+ cell population in the upper left. While the “Cell Health (Calcein AM Viability)” app makes this analysis easy and straightforward, any app that uses both fluorescent channels will work well (like the “GO Flow” app used for the AO/PI data seen above).
Summary
A variety of staining options exist when assessing the viability of a sample. If doing single-color viability stains using PI, both the Moxi V and Moxi GO II will provide unparalleled accuracy for both counting and viability on more cells per sample compared to the competition. If extra levels of viability measurements combined with PI are needed, the Moxi GO II is capable of detecting any additional green fluorescent dyes and is the instrument to choose.