In the previous section , we focussed on the use of flow cytometry specifically for immunophenotyping. However, flow cytometry can be used for a vast array of purposes by research scientists and clinicians. In this section, we will look more broadly at some of these possible applications of flow cytometry, new technologies that utilise flow cytometry and look to the future to discuss emerging applications.

Assessment of Apoptosis Markers

Flow cytometry can be used to rapidly quantify markers of cell death. Measurement of cell viability is important for almost all flow cytometry experiments; dead cells can confound analysis of antibody staining. Viability of living cells can easily be assessed by adding DNA binding compounds. These probes intercalate with DNA, but will only have access to DNA in cells with dam aged membranes, in other words during death. Discrimination of live and dead cells is mostly frequently undertaken using 7-AAD, propidium iodide or DAPI. Furthermore, there are now a number of commercially available fixable live/dead stains, which allow discrimination of live and dead cells in tandem with, for example, intracellular cytokine staining. Antibody stains are also available to assess cell survival factors and other hallmarks of apoptosis, such as active caspase 3 or Bcl-2.

Detection of Changes to Surface Membrane

An early change during apoptosis is translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Ultimately, the integrity of the plasma membrane is compromised, but early in the process, only phosphatidylserine is exposed. The calcium-dependent membrane binding protein annexin A5 possesses substantial specificity for phosphatidylserine; fluorescently labelled annexin A5 is thus able to stain phosphatidylserine on the surface of apoptotic cells. In tandem with a cell viability dye, such as 7-AAD, it is possible to detect annexin A5 positive and 7-AAD negative cells, which represent early apoptotic cells with an altered plasma membrane. In contrast, a simultaneous positive staining with annexin A5 and 7-AAD indicates dead cells.

Functional Assays

There are a number of assays that can be undertaken with flow cytometry to assess cellular functions. These can be combined with detailed cellular phenotyping to provide comprehensive datasets on cell populations present in blood and tissues. A few of the most commonly employed assays will be discussed in the following.

Calcium Flux Analysis

 A key cellular function that can be assessed by flow cytometry is calcium signalling, a downstream signal in multiple activation pathways. Through use of calcium-binding dyes, intracellular calcium influx can be assessed in cell populations following stimulation by means of establishing baseline intracellular calcium levels, as well as the kinetics (magnitude and duration) of the calcium flux.

Indo-1 is the most commonly employed dye in this context. Its excitation wave length is in the ultraviolet region (λ_exc = 346 nm) and it possesses distinct emission spectra depending on whether it is in its free form ( λ_em = 475 nm; green) or calcium-bound (λ_em = 401 nm; violet). Typically, it is the ratio of intensities at both wavelengths that is monitored over time (so-called ratiometric technique ). Prior to activation, the signal will be predominately green; upon cell stimulation, and release of intracellular calcium, the dye molecules will bind Ca 2+ ions and emit at the violet wavelength. Indo-1 can be employed to assess calcium-signalling kinetics in a wide variety of cell types; all that is required is an agonist that will illicit a calcium flux in the cell type of interest. For example, activation of the T cell receptor on lymphocytes can be employed to assess T cell calcium flux. Key control experiments include positive and negative stains to ensure Indo-1 loading and emission. Most frequently, the calcium ionophore ionomycin is used as a positive control, generating the maximum calcium release into the cytoplasm and therefore greatest emission of Indo-1 in the calcium-bound state. In contrast, calcium chelators, which will blunt calcium signals, are employed as negative controls.

Practically, Indo-1 is used to stain a sample and the loading of cell populations is assessed on the flow cytometer in order to allow a baseline of Indo-1 fluorescence to be established. Without halting data acquisition, the sample tube is briefly removed from the SIP and the cell stimulant is added to the tube. The tube is then returned to the SIP and ratiometric Indo-1 emission is followed against time, as shown in Figure1.

Fig1. Calcium flux analysis via flow cytometry. Flow cytometry plots (in the absence and presence of additional calcium in the media) show ratiometric change in Indo-1 fluorescence following activation of calcium signals. By monitoring the change in fluorescence ratio for Indo-1 against time, a calcium baseline, calcium signalling kinetics and magnitude can be determined, as indicated.

Phosflow

 Another method that can be employed to query cellular function by flow cytometry is phospho-flow cytometry, or ‘Phosflow’. Here, intracellular signalling pathways are examined by assessing phosphorylation of protein sites on key signalling proteins. By using antibodies against specific phosphorylated proteins, Phosflow can be employed to measure signalling cascades in individual cells. This method is therefore not merely an alternative to Western blotting, but a complementary technique to this staple method of assessing cellular signalling pathways, as it offers a reliable way to measure signalling events in individual cells. Western blotting, by contrast, measures the average level of phosphorylation within the entire population. A disadvantage of Phosflow in this context is that it is not possible to determine the relative molecular mass of the targeted protein – information that can be useful to confirm the specificity of the antibody.

Practically, cells are stimulated and then fixed using paraformaldehyde-based buffers allowing the induced states of protein phosphorylation to be preserved. Next, cells are permeabilised and then stained with fluorescently conjugated antibodies against the phosphorylated proteins of interest. This can occur simultaneously with antibodies against surface markers, allowing cellular signalling events to be assessed in multiple populations of cells simultaneously. For example, Phosflow can be used to assess mitogen-activated protein kinase (MAPK) signalling events or mechanistic target of rapamycin (mTOR) signalling. The bottleneck in assessment of signalling pathways in this context remains the generation of phospho-specific antibodies that work in cells that have been fixed with paraformaldehyde.

Cell Proliferation

 There are a number of ways to assess cellular proliferation by flow cytometry. Here, we will discuss the most commonly employed, which allow assessment of cell proliferation in different ways. Alongside the applications outlined below, straightforward flow cytometry staining for the intracellular proliferation marker Ki-67 can also be conducted to assess cell proliferation. Ki-67 is an antigen that is only exposed during interphase, and therefore positive staining for Ki-67 marks proliferating cells.

• Cell cycle analysis: DNA-binding dyes such as propidium iodide are stoichiometric; as more DNA is present in the cell it will bind more dye, resulting in greater fluorescence emitted by propidium iodide. This can be exploited to determine the current phase of the cell cycle of the examined cells. Specifically, cells in S phase will have more DNA than cells in G1 phase; furthermore, cells in G2 phase should be twice as bright as those in G1 phase, as they contain double the amount of DNA ( Figure 2a).

• Carboxyfluorescein succinimidyl ester (CFSE) dilution: CFSE is a fluorescent dye that covalently binds free amine groups present on intracellular molecules via its succinimidyl group. This conjugation leads to incorporation of CFSE within the cell and prohibits transfer of the dye to other cells. With this approach, the proliferation of the CFSE-labelled cells can be monitored. Each time the labelled cells divide, the CFSE fluorescence emission will be halved, with the daughter cells receiving half the amount of CFSE-labelled proteins. In this way, the number of cell divisions over time can be determined from the CFSE dilution within a population of cells (Figure 2b). This method can typically trace up to eight generations of cell division.

Fig2. Examination of cell proliferation by flow cytometry. Two common assays employed to assess cell cycle progression and cellular proliferation are outlined. (a) The plot shows staining for propidium iodide on gated cells to assess cell cycle progression. Intensity of propidium iodide staining indicates whether the cells are in G0/G1, S or G2/M phase. (b) This plot shows cell proliferation assessment based on staining for CFSE. The intensity of CFSE staining decreases as cells proliferate, since half the amount of CFSE is passed on to the daughter cells. As such, the number of times a cell has divided can be monitored by examining how much the CFSE fluorescence has been diluted.

Flow Cytometry Combined With Other Technologies

Flow cytometric technology has been coupled with other techniques to develop novel applications for use in research and also the clinic. A few of these will be outlined in this section and demonstrate the versatile use of flow cytometry throughout the scientific world.

ImageStream ®

Imaging with flow cytometry combines flow cytometric analysis with microscopy, allowing specific cell populations to be identified by conventional flow cytometry, but, at the same time, imaging each cell. These specialised flow cytometers take high-resolution images of each cell. The images can be bright-fi eld images to assess morphology or enhanced with fluorescence information. Experimentally, this method allows assessment of protein localisation within a cell alongside examination of cell morphology and the usual benefits of flow cytometry. Although generating large data fi les, employing an ImageStream ® allows for a substantial number of cells to be examined, providing statistical assessment of cellular phenotypes, as well as generating good-quality fluorescence images of the cells.

Multiple Analyte Detection Systems

 Often referred to as cytometric bead arrays, this application of flow cytometry allows an array of soluble proteins to be analysed simultaneously. This technology employs fluorescently labelled beads that are coated with a capture antibody targeting a specific analyte, most commonly a cytokine. Multiple analytes can be analysed simultaneously, as beads coated with different capture antibodies can be distinguished from each other based on fluorescence. By employing cytokine standards, similar to those used in ELISAs, concentrations of analytes in serum, tissues or cell culture can be determined. The key advantage of this application over more traditional methods such as ELISAs is that multiple analytes can be simultaneously quantified using as little as 25–50 μl of sample.

Uses of FACS

As well as sorting highly pure populations of cells for further analysis, FACS is also a key experimental tool with a broad array of applications. The ability to separate cell populations based on specific markers is invaluable for research and a few of the FACS applications are discussed below:

• Enrichment of transfected cells: Importantly, FACS is the only method for cellular purification that is able to separate cell populations based on internal staining. For example, FACS can be employed to sort GFP-expressing cells. The applications of this advantage would allow, for example, transfected (and therefore GFP-positive) cells to be separated from non-transfected cells.

• Genomic technologies and single cell examination: The ability to isolate highly pure populations of cells has heralded the onset of in-depth genomic assessment of these cells. Microarray analysis and RNA-sequencing ( RNA-Seq) are frequently coupled with FACS to examine the transcriptome of the sorted cells. In the immunology fi eld, the ImmGen consortium aims to probe the transcriptome of all immune cells in this way. Through the use of established as well as novel surface markers for cell populations, very specific populations are isolated and examined, providing detailed information on gene expression and gene networks in immune cells. Building upon this application, more recently, groups have begun to isolate individual cells and subject these to next-generation sequencing technologies. Importantly, FACS can be employed to isolate single cells in a high-throughput manner, allowing many single cells to be obtained for further genomic examination. This approach allows the heterogeneity of cell populations to be resolved at a transcriptional level, providing a better understanding of the cellular response of the isolated cell population.

• Ig-sequencing; ‘Bug FACS’ : As well as being employed to assess cell populations, phenotype and functional attributes, flow cytometry can also be utilised to query interactions between the immune system and the commensal bacterial communities that cover our bodies. These microbial communities, termed the microbiome, are key for full maturation of the immune system, but full elucidation of the extensive and intricate interactions between these microbes and the host immune system is outstanding. One way to assess these interactions is to determine which microbes have been encountered by the immune system and subsequently initiated the production of microbe-specific immunoglobulins (Igs). By employing flow cytometry, bacterial communities, from any site in the body (including the gastro intestinal tract, saliva or oral biofilm) can be examined to see which bacteria are recognised, and therefore bound, by Igs. Antibodies against different Igs, for example immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG), can be used to ascertain the type of immune recognition driven by the Ig-bound bacteria. Through use of cell, or in this case bacteria, sorting, the Ig-bound bacteria can be separated out from the non-bound bacteria and subjected to deep-sequencing techniques such as 16S-sequencing, providing an assessment of bacteria capable of driving antibody responses at specific sites.

Translational Applications in Clinical Practice

 Flow cytometry is a routine tool used by clinical pathologists and immunologists to diagnose patients with certain types of cancer or immunodeficiencies. There are novel patterns of surface-marker expression on certain cancers and unique pathophysiologies that, when analysed via flow cytometry, lead to the determination of specific treatment strategies. In addition to being employed as a diagnostic tool, by utilising FACS it is possible to isolate tumour-free populations of stem cells for stem cell transplantation. Flow cytometry also provides a rapid method for looking at cytokine production and HLA genotypes, and as such the translational applications of flow cytometry are considerable.

Cancer Diagnosis and Prognosis

Flow cytometry can be used to help identify the distinct immune phenotypes of malignancies and also cell DNA content, both of which can be used to help guide selection of therapies. One of the best examples is in CD20+ B cell malignancies that are now commonly treated with anti-CD20 antibody (rituximab). Flow cytometry can be used to distinguish between B cell malignancy and more aggressive lymphomas, such as mantle cell lymphoma. This is done by looking for a reproducible expression pattern of multiple antigens or antigen density on malignant cells. Additionally, because flow cytometry can detect individual changes in cells, even in a heterogeneous population, it can also be employed to identify residual malignant cells following therapy. This is termed minimal residual disease monitoring as, frequently, malignancies in which there is disease persistence eventually result in worse disease outcome.

DNA Content Analysis

Relative cell DNA content can easily be measured by flow cytometry and this can be used to help guide treatment in acute lymphoblastic leukaemia . As described, DNA content can be measured by employing a fluorescent dye such as propidium iodide that intercalates with DNA. The fluorescence emission is directly proportional to the amount of DNA in the nucleus and so it can be used to establish gross gains or losses in DNA. This can be used in a tumour cell population to establish abnormal DNA content or ‘ DNA content aneuploidy’. This is typically associated with malignancy; however, it can also occur in certain benign conditions. DNA aneuploidy can be associated with worse or better outcomes, depending on the type of cancer.

Immunologic Diseases

A major use of flow cytometry is to determine CD4+ T cell counts following HIV infection. It can also be used to classify and assess prognosis of various primary immune deficiencies, including severe combined immune deficiency (SCID), auto immune lymphoproliferative syndrome and antibody deficiencies. Additionally, flow cytometry can be used in allogeneic stem cell grafts to look at specific immune cell populations and also assess immune reconstitution following allogeneic bone marrow transplant to predict survival post-transplant. Flow cytometry is similarly employed to look for rare CD34+ endothelial cell progenitors that can help to predict clinical outcome in patients that have peripheral artery and cardiovascular disease.

Cellular Transplantation

FACS can be used to enrich for specific cell populations that are required for cell based therapy, such as, for example, CD34+ human hematopoietic stem and pro genitor cells. In essence, autologous CD34+ stem cells can be sorted to high purity with the possibility of achieving a clinically significant depletion of contaminating tumour cells. Studies with patients suffering from non-Hodgkin’s lymphoma, breast cancer and multiple myeloma provided the proof of concept that durable hematopoietic reconstitution can occur and robust remission achieved when these highly purified cells are transferred.

Flow cytometry is also frequently employed in the clinic to measure antibodies to human leukocyte antigen (HLA) molecules. Individuals generate antibodies to these molecules, for example following exposure during pregnancy or blood transfusion. The presence of donor-directed anti-HLA antibodies poses a significant risk of allograft rejection. Flow cytometry provides a high-throughput method to determine the presence of antibodies to HLA in patients due to undergo transplantation. HLA genotyping can also be performed by flow cytometry by employing a multiplexing platform. Here, high throughput examination of HLA-genotypes can be determined in samples derived from patients and donors undergoing transplantation.

The Future of Flow Cytometry

 Since its advent, multi-colour flow cytometry has advanced significantly, marked in part by the expansion of the historic two-colour flow cytometry to the 20+ colours that are detected in some settings today. Moreover, the technology has advanced in such a way as to allow not only more detailed and complex flow cytometry panels to be assessed, but also to make instruments available that are smaller, simpler to use and less expensive. Further features include more automated packages and, in some cases, even portable instruments, thus widening the user base and applications for this technology.

The continuing evolution of this technology will involve the ability to visualise and differentiate between even more tagged antibodies. Although there may be a maximum number of fluorescent labels that can feasibly be used, there are other ways to tag antibodies. Mass cytometry , or CyTOF, allows visualisation of antibodies labelled with heavy metal ions instead of fluorescent labels. Whether a cell is labelled with an antibody or not is determined by time-of-flight analysis, similar to mass spectrometry. This new methodology overcomes the issues of spectral overlap between fluorescent dyes, allowing many more differentially labelled antibodies to be employed.

In line with the expansion of the technology, analysis packages have also developed. Moving forward, a clear advancement on most of the currently employed analysis packages will be the development of automated systems for data analysis. Whereas automation exists to a certain degree, the ability to assess multiple experimental variables in continuously changing staining panels is certainly a requirement for further advancements. Similarly, increased abilities of automated data interpretation will enhance the high-throughput applicability of this technology. Clearly, flow cytometry promises to continue to be used in a vast number of different arenas in the years to come, as it is today.

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طريقة مبتكرة للكشف المبكر عن أمراض القلب والشرايين

منظمة الصحة تحذر من خطر إدمان الشباب لأكياس النيكوتين

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الآخبار التكنلوجية

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دراسة حديثة تفند ربط التستوستيرون بالمخاطرة لدى الرجال

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المزيد

مواضيع ذات صلة

?Why use a Radioisotope

Flow Cytometry: Practical Considerations

Fluorescence Labels

Fluorescence-Activated Cell Sorting (FACS)

Flow Cytometry – Instrumentation

Antibody Preparation

Harnessing the Immune System for Antibody Production

Electrophoretic Techniques : Support Media and Buffers

Principles of Liquid Chromatography

Preliminary Purification Steps

Cell Disruption and Production of Initial Crude Extract

Determination of Protein Concentrations