Fluorescent Cell Testing Measures – Assessing cell proliferation and cell health using flow cytometry and imaging platforms

Introduction

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.

Are my cells alive? Are my cells dividing and proliferating? Are my cells healthy? Are you having issues with cell cycle measurements? In this ask-an-expert session, we invite you to ask questions around fluorescent testing measuring cell proliferation and assessing cell health using flow cytometry and imaging platforms.

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Question 1

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

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 … Continued

Question 2

What methods do you think are best for imaging of non-adherent cells?

Imaging of non-adherent cells can be difficult, mainly because they don’t image very well. Here are some things to consider: • confocal imaging will help since non-adherent cells tend to be spherical • if labeling intracellular structures, washes and optimization is really important since the internal cellular architecture is not as nicely spaced out like … Continued

Question 3

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?

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 … Continued

Question 4

when measuring dna content histograms in flow cytometer with DAPI, I often observe a shift of the whole cell cycle distribution to the left or to the right when changing from one sample to another. why is this, how can I avoid this effect? my samples are all treated the same way (same culture method, same fixation and permeabilisation method etc).

DNA content cell cycle analysis requires careful optimization of every step in the process. Under ideal staining conditions, all cells with the same DNA content are expected to be uniform in staining. However in practice, variation can result from differences in sample prep, staining, methods of acquisition and anaylsis, along with performance of instrumentation. For … Continued

Question 5

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

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. … Continued

Question 6

I have cells expressing GFP and I want to use CSFE, but they both are green-emitting. Can you suggest something?

There is another popular dye, CellTrace Violet, which uses violet excitation with blue emission. CellTrace Violet is fully compatible with GFP. CellTrace Violet and CellTrace CSFE are both amine-reactive succinimidyl ester compounds that covalently bind to proteins and give a bright homogenous fluorescence. When cells divide, the fluorescent label is distributed equally between daughter cells. … Continued

Question 7

What are the advantages of live cell imaging and would you use live-cell incubation chambers or another method?

Live cell imaging has transformed the way biologists study cells, proteins and a variety of processes and molecular interactions. Live cell imaging techniques allow the observation of internal structures and cellular processes in real time, and across time. Understanding cellular structures and dynamic processes can be critical in the study of cell biology. The observation … Continued

Question 8

How can you ensure that your media choice won’t interfere with your imaging? Are there certain components to avoid?

Phenol Red can cause problems with imaging, it is best to use a media that does not have phenol red in the formulation. One challenge with live cell fluorescence imaging is in imaging weak fluorophors without causing cell damage, photobleaching, or changes to cell health. A newer media from Gibco is FluoroBrite DMEM; it is … Continued

Question 9

We had a culture crash and a large portion of cells died. Is there any way to use imaging to try and discover the cause.

Some general causes of cell death include contamination from infections (yeast, bacteria, mycoplasma, viruses), chemicals (sterilizing solutions like ethanol or bleach), or issues with the cell line or methods of culturing. To establish and successfully maintain cell cultures requires standardized approaches for media preparation, feeding, and passaging of the cells. Cultures should be examined regularly … Continued

Question 10

What can I do if when using flow cytometry I can’t get to running my samples right away. What can I do to still get good staining?

Often there is a delay between sample prep+staining before acquisition on the flow cytometer. Depending on the assay, the stained cells may be fixed and data acquired within 24 hours, but not all assays can be fixed in this manner. Some assays require the cells be fixed as part of the sample prep and staining, … Continued

Question 11

How can I tell the difference between alive and dead cells during flow cytometry?

You can use fluorescent labels to identify dead cells, to identify live cells, or combine them both in a two parameter assay. The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide or a dye from the STYOX series. A healthy living cell has an intact … Continued

Question 12

What is the best way to look at intercellular events?

What you are testing for will determine the best method for detecting intercellular events. If your target of interest requires spatial resolution, imaging is the best platform to use. The cells will need fixation and permeabilization before labeling. The technical data sheet for the fluorescent product will list this out. If you are looking for … Continued