Flow cytometry as a tool for the investigation of PI3K-Akt-mTOR signaling in AML 43

We used flow cytometry, a laser-based analysis technique, for the study, characterization, and detection of the cells. A flow cytometer performs simultaneous and rapid quantitative measurements of numerous physical and chemical characteristics.

The basic principle behind the method is the passage of particles in single file in front of a laser, which detects, counts, and sorts them. The cells are fluorescently labelled using antibodies and are excited by the laser though the emission of light at different wavelengths. The fluorescence can then be measured to determine the type and quantity of cells present in the given sample.

Prior to measurement, particles in suspension are hydrodynamically focused to maintain proper alignment and separation of the cells within the fluid stream. The stream is run through one or more lasers, resulting in the detection of florescent and scattered light by photomultiplier tubes (PMT) (Figure 7). The signal is then amplified and converted to a voltage pulse and finally transformed to a digital value. While the run of each sample includes measurements of a large number of cells, the strength of the method is that each cell is measured by itself, allowing for the identification and grouping of individual cells.

The technique uses fluorescent reagents, usually monoclonal antibodies, for the identification and stratification of cells.

Fluorochromes are excited at a specific wavelength of light provided by the laser and emitted at a lower specific wavelength. Emitted light of a specific wavelength is distinguished by the use of suitable optical filters prior to the detection of the emitted light. This increases the number of parameters that can be detected.

Figure 7. Schematic of the basic steps of flow cytometry. A) A heterogeneous cell sample can be stimulated to prompt specific signaling cascades and phosphorylation of the proteins of interest. The cells are fixed, permeabilized, and stained with fluorophore-conjugated for identification of cell type and mediator of interest. The cells are then analyzed on a flow cytometer. B) A cell suspension is hydrodynamically focused to intersect with a laser. The signals from the laser are collected by a forward light scatter detector, a side light scatter detector, as well as various detectors for fluorescence emissions. Finally, the signals are amplified and transformed into digital data for further analysis.

4.6

Intracellular flow cytometry

The main method used in these studies is flow cytometric detection of total protein and protein phosphorylation, and all experiments (article I-IV) were performed on a FACS Verse flow cytometer. Flow cytometry offers several benefits for studies investigating cell signaling based on its ability to assemble data at the individual cell level. Detection of intracellular signaling responses mediated through protein phosphorylation for defined cell subsets within a complex cell population can be accomplished through the combined use of fluorescent subset-identifying antibodies (e.g. detection of specific cell surface molecules) and antibodies specific to protein phosphorylation sites.

The processes that occurs within a cell are important for many aspects of cell biology and medicine. The understanding of the function of normal cell metabolism, cell response to environmental factors, and eventually, cell aging and cell death, can provide insight into how diseases develop and functions. More importantly, knowledge of how

and when these normal processes malfunction is essential for the understanding of disease and the development of more effective treatments and cures. The ideal method of analysis would be to study and obtain information from living cells, without the need for labels. Instead, cell fixation is often used to allow a snapshot of a cell state to be measured. To detect specific phosphorylated proteins using flow cytometry, cells have to be fixed to preserve the phosphorylated state of the signaling proteins, and then the cell membrane must be permeabilized to allow antibodies to enter the cells and bind to their target epitopes.

Aldehydes, including paraformaldehyde, function as cross-linking agents that react with proteins and nucleic acids within the cell, and are widely used in fixation. They provide good preservation of the cellular structure and their use does not appear to result in significant structural changes to the proteins.

Following paraformaldehyde, methanol was used for permeabilization in all our studies (article I-IV); and cells were then stained with phosphospecific antibodies. Organic solvents, such as methanol, acetone, and ethanol preserve both cell morphology and nucleic acid content [207, 208]. Moreover, these fixatives have been shown to be more effective for the preservation of nucleic acid content in cells than aldehyde-based fixatives [208]. Methanol combined with low non-specific staining has the ability to increase the reactivity of antibodies to certain nuclear antigens [209], and cold methanol is often used as a permeabilization agent when flow cytometry is used to detect phosphorylated proteins and transcription factors. However, one of the potential problems with methanol is the denaturation of surface receptors [207, 208]. We tested our surface markers for studies I-IV before and after treatment with methanol and detected the same markers at both times.

Protein phosphorylation is temporary and is regulated by protein phosphatases. Most methods for detection of protein phosphorylation require prompt inactivation of phosphatases immediately following stimulation. All cell samples were therefore swiftly fixed to sustain phosphorylation. In order to avoid unspecific antibodies binding to cells, the antibodies must be titrated. The degree to which this is a problem may differ between different antibodies that are specific for the same antigenic epitope. We used the same

well-characterized antibodies throughout all of our studies to avoid the problem of variation in nonspecific binding between different experiments. During titration testing, we also found that the most effective concentration of antibodies for a staining mixture was essentially independent from the number of cells being stained but strongly dependent on stain volume. Hence, the same stain volume was used in all experiments.

The normal level of cell fluorescence, referred to as autofluorescence, may present an issue for data analysis of flow cytometry. Mammalian cells contain many compounds that are excited by the 488 nm laser that is commonly used in flow cytometry. Hence, the signal to noise ratio is reduced resulting in a decreased sensitivity and more false-positive cells. Spillover refers to the overlap among the emission spectra of certain commonly used fluorochromes, and spillover from one channel to another may mask low expressers. This can be corrected using unstained controls, and fluorophores that emit above 600 nm will have less autofluorescence interference. Fluorophore brightness is a relative indication of the intensity of a fluorophore, measured above the background (dim=1 to bright=5). The use of a bright fluorophore will diminish the effect of autofluorescence. In addition, we stained cells separately with each antibody as a compensation control.

Cell signaling studies often investigate cellular responses to treatment with stimulatory or inhibitory molecules, making unstimulated or untreated cells the best controls for evaluation of background staining. In the past, isotype controls were widely used to correct for unspecific binding of antibodies. However, an unstimulated cell control considers the background characteristics of each antibody as well as the basal phosphoprotein expression levels within the cells of interest, which is different from an immunoglobulin isotype control. For this reason, we included three unstimulated cell controls for all flow cytometric assays in articles I-IV. For stimulation studies, we also included unstained controls with each stimulus or inhibitor, as well as stimulus plus inhibitor controls.