Lesson 8: Fluorochromes

It is now time to take a closer look at Experimental Design. The choice of markers (antibodies, dyes, etc) and fluorochromes go hand in hand. However, the choice of antibodies is generally dictated by previous work (literature search, ongoing research, etc), and it is when choosing fluorochromes that flow cytometry-specific questions need to be answered. Thus we will focus our attention on how to make intelligent fluorochrome choices.

The Decision Tree

Research BRCF Flow Cytometry Training Decision Tree for Fluorochromes

While fluorochrome choice may seem, at first, to be the least of decisions when designing an experiment, as you can see from the process tree above, it is far from simple. Many things must be considered when pairing markers with fluorochromes. In order to simplify the process, lets look at the above tree one piece at a time.

Reagent Availability

Research BRCF Flow Cytometry Training Reagent Availability of Fluorochromes

The first item to look at is reagent availability.

  • Antibodies – Will you use conjugated antibodies as we discussed in lesson 5? Are there directly-conjugated antibodies available from a commercial or in-house source or will you need to use a secondary antibody staining such as the Streptavidin/Biotin combination we discussed previously? What fluorochromes (Fitc, PE, APC, etc) are used in the available reagents? Is there a different fluorochrome available if each individual marker (or group of similar-function markers)? If you are using a secondary system, will you be able to limit secondary staining to a single marker or will you need to consider this type of two-step staining for multiple markers? (Please note that while multiple secondary stains are possible, such systems are prone to noise and inaccurate signal measurement).
  • Stains – If your experiment includes chemical stains (such as Propidium Iodide, a fluorescent dye we’ve already discussed), what is the excitation and emission spectra of the reagent? Will it interfere with any previously chosen reagents? CFSE, for instance, is a green stain and would be detected in the same channel as Fitc. Therefore, if CFSE is to be used, Fitc should be avoided.
  • Other – Remember that any source of cellular fluorescence needs to be factored in to your experiment. Perhaps the cells have undergone viral transfection to insert a new gene. Co-transfection with GFP (Green Fluorescent Protein) would allow for those cells which have successfully incorporated the new gene to produce green fluorescence under standard 488nm laser excitation. Of course, as a green signal, Fitc should, once again, be avoided.

Instrumentation Availability

Research BRCF Flow Cytometry Training Fluorochromes Instrumentation availability diagram

Next, one must consider the instrumentation available to the investigator. A useful chart of common instrument configurations and typical fluorochromes is available here.

  • Excitation Sources – What excitation sources (lasers, arc lamps, other) exist on the available flow cytometer? Are the light sources fixed in wavelength or frequency-agile? Are the available excitation sources capable of sufficiently exciting the tentative fluorochromes?
  • Optics – What filter/detector combinations are available on the optical deck of the cytometer? Are the available optics capable of resolving the tentative fluorochrome list or will additional optics need to be purchased (if available)?
  • Compatibility – Finally, some thought needs to be made as to whether the chosen fluorochromes are compatible with the existing equipment. As an example, if a chosen fluorochrome is prone to Photo-Damage (such as PerCP), it is best reserved for cytometers utilizing low-power laser systems. Other fluorochromes may produce a very weak signal which is best detected via closed flow cell systems rather than less sensitive stream-in-air systems.

Details of available core instrumentation and available excitation sources can be found on the Flow Core web site. Beginning investigators are limited to self-operation of the FACSCalibur and XL analyzers.

Practicality

Finally, after considering available reagents and instrumentation, one must consider whether the available staining protocols are practical in the lab. Some things to be considered include:

  • Cost – Unfortunately, scientific work, like everything else in this world, must bend to budgetary constraints. Is the tentative experimental plan cost effective?
  • Timing – Many staining procedures have dictated time spans. Whether it is a required pre-incubation prior to staining, incubation time during staining or a short fluorochrome lifetime, it is necessary to look at the experiment from a holistic point of view and determine whether the necessary time and technicians are available. Will there be viability issues?
  • Hazards – Not all reagents are innocuous? Will the procedure introduce unnecessary risks for the technicians performing the experiment? Will the procedure introduce an unanticipated potential pathogen. As an example, Lentivirus, an HIV derivative, is a fairly innocuous virus with which to work. However, when co-transfected, it is prone to recombination. Will your procedure cause the lenti to become replication-competent?
  • Instrumentation Damage – Will the chosen protocol damage the instrument itself? Acridine Orange, for instance, is a useful cell cycle marker, but is prone to permanently staining the cytometer flow cell and tubing when used improperly. Is the owner of the cytometer willing/able to re-tube the instrument if necessary?
  • Cell Toxicity – Finally some markers provide very useful data, but are toxic to the cells. Hoescht stains, for instance, have a high-level of toxicity. Therefore, if the experiment involves collection and reuse of the cells (perhaps for animal injection or cell culture work), this stain is best avoided.

As stated earlier, fluorochrome choice is far from simple. However, by following a careful decision process, it needn’t become a burden.

Wrapping Up
In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.

  • Using the following chart and your t-cell/b-cell enumeration protocol from lesson 5, determine if the previous fluorochrome selection is compatible with the FACSCailbur. If not, adjust your protocol accordingly.
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