We recently finished our Ask the Expert discussion on assessing cell proliferation and cell health using flow cytometry and imaging platforms. This week we had many interesting questions and valuable answers and suggestions. Flow cytometry topics included detecting dead cells, DNA content, and timelines for running samples. In cell imaging, questions areas included using imaging to look at intercellular events, improve productivity, maintain cell health and viability, and determining causes of cell culture crash. Other subjects included media selection for best imaging, dye selection, imaging non-adherent cells, live cell imaging and equipment, fixation methods and propidium iodide for dead cells and cell cycle.

Cell Proliferation assays are an important set of fluorescence based tests that can monitor cell health, cell division, and cell proliferation using a variety of techniques involving flow cytometry and imaging platforms. From DNA content cell cycle, to tracking of generational cell division, to simple viability and vitality measurements, there are assays that can provide a rich data set to answer simple or complex questions and provide direction for future experimentation.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Jolene Bradford, R&D Associate Director in Eugene, Oregon, USA working in the Biosciences Division of Thermo Fisher Scientific (formerly Life Technologies). Jolene joined Molecular Probes® Labeling and Detection Technologies in 2001, where she has developed new reagents and assays for the flow cytometry platform. Well versed in fluorescent assays to evaluate proliferation and cellular health, Jolene has provided numerous webinars and seminars on the topics. She has been an invited instructor at many flow cytometry training courses and workshops, both in the US and abroad. More recently she has been involved in developing acoustic cytometry. Prior to joining Molecular Probes, Jolene spent over 20 years in healthcare performing clinical laboratory testing as a specialist in hematology and flow cytometry.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – assessing cell proliferation and cell health using flow cytometry and imaging platforms

I’m doing cell cycle in fixed cells. Does the method of fixation matter?

The Answer:
Fixation has two functions. First, it preserves the cells by preventing lysis and autolytic degradation. Second, it makes the cells permeable and thus their DNA accessible to impermeant DNA-binding dyes.

Precipitating fixatives (methanol, ethanol, acetone) are preferred for single color cell cycle staining. Disadvantages include some epitpoes are destroyed and there is more cell aggregation.

Crosslinking fixitves (-aldehydes) can have a harmful effect on stoichiometry of DNA staining, but fewer epitpoes are destroyed and less cell aggregation is induced. Although inferior in terms of stabilization & preservation of low molecular weight constituents within the cell, they adequately stabilize undamaged DNA.

You will need to choose the best fixation method for your application. Or you may use a cell-permeant dye such as the Vybrant DyeCycle stains or UV-excited Hoechst 33342 for cell cycle in living cells.

I don’t understand how PI can be used for dead cells and for cell cycle.

The Answer:
Propidium Iodide (PI) is a cell impermeant DNA binding dye. In a population of cells, there are live cells and dead cells, and PI can be used to identify dead cells in a mixed population. In this case a healthy cell membrane will exclude the dye; the cell membrane forms an intact barrier to exclude the dye from getting into the cell. When a cell is dead, the cell membrane is compromised, and the PI dye is allowed inside the cell where it can bind to the DNA and become fluorescent, and thus identify the dead cells. This uses a log scale on your flow cytometer.

When cells are fixed, PI can be used to determine DNA content cell cycle. During fixation, the cell membrane loses its integrity and so PI may freely enter the cell to bind to nucleic acids. Because PI is not DNA-selective, remember to add RNase to your staining solution. This uses a linear scale readout on your flow cytometer.

We are looking to improve our cell culture productivity in CHO cells. We want to use imaging to inform our process choices and improve our cell health, viability and of course titer. What methods would you recommend?

The Answer:
There are a number of choices for evaluating the health of your cells using fluorescent imaging assays, from simple to complex. Understanding how healthy your cells are, and determining optimal timing for passage can improve productivity. A number of assays can help provide useful information.

• To evaluate viability, the use of a cell-impermeant DNA binding dye may be used, such as the Image-iT DEAD Green Viability stain where dead cells will fluoresce green. A dead cell stain can be combined with a marker of cell vitality that measures esterase activity, Calcein AM, as seen with the LIVE/DEAD Cell Imaging Kit where active cells fluoresce green and dead cells fluoresce red. Probably the most simple method uses ReadyProbes Dead Cell stains; these are ready-to-use reagents for fluorescent imaging that have been simplified for use (add 2 drops from the dropper bottle per mL of sample and image).
• Cell proliferation analyses can be helpful for assessing cell growth. A very useful method uses incorporation of a thymidine analog, EdU, for direct measurement of cells in S-phase using click chemistry. The Click-iT Plus EdU assays are fully compatible with fluorescent proteins.
• You may want to know if your cells are dying via apoptosis, the CellEvent Caspase 3/7 Green reagent is a no-wash assay compatible with labeling in media, the green fluorescence intensity increases with caspase activity.