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In cancer therapy, immunogenic cell death eliminates tumor cells more efficiently than conventional apoptosis. During photodynamic therapy (PDT), some photosensitizer (PS) targeting lysosomes divert apoptosis to the immunologically more relevant necrosis-like cell death. Acridine orange (AO) is a PS targeting lysosome. We synthesized a new compound, 3-N,N-dimethylamino-6-isocyanoacridine (DM), a modified AO, aiming to target lysosomes better. To compare DM and AO, we studied optical properties, toxicity, cell internalization, and phototoxicity. In addition, light-mediated effects were monitored by the recently developed QUINESIn method on nuclei, and membrane stability, morphology, and function of lysosomes utilizing fluorescent probes by imaging cytometry in single cells. DM proved to be a better lysosomal marker at 405 nm excitation and lysed lysosomes more efficiently. AO injured DNA and histones more extensively than DM. Remarkably, DM's optical properties helped visualize shockwaves of nuclear DNA released from cells during the PDT. The asymmetric polar modification of the AO leads to a new compound, DM, which has increased efficacy in targeting and disrupting lysosomes. Suitable AO modification may boost adaptive immune response making PDT more efficient.

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Briefly depicted are the publications in CYTOMETRY that received the highest frequency of citations. Among them are seminal papers describing application of metachromatic fluorochrome acridine orange to differentially stain DNA versus RNA or to analyze susceptibility of DNA in situ to denaturation; both features being markers of different sections of the cell cycle including identification of noncycling quiescent cells. The papers reviewing detection of cyclins D1, E, A or B1, each in relation to cell cycle phase, were also among the highly cited ones. The highest citation rates received publications describing development of the TUNEL methodology to detect apoptotic DNA fragmentation, and more recently expression of ϒH2AX to reveal DNA damage. © 2020 International Society for Advancement of Cytometry.

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Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

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Activation of caspases is a characteristic event of apoptosis. Various cytometric methods distinguishing this event have been developed to serve as specific apoptotic markers for the assessment of apoptotic frequency within different cell populations. The method described in this chapter utilizes fluorochrome labeled inhibitors of caspases (FLICA) and is applicable to fluorescence microscopy, flow- and imaging-cytometry as well as to confocal imaging. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine, when applied to live cells in vitro or in vivo, exclusively label the cells that are undergoing apoptosis. The FLICA labeling methodology is rapid, simple, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. FLICA staining followed by cell fixation and stoichiometric staining of cellular DNA demonstrate that FLICA binding can be correlated with the concurrent analysis of DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and therefore more accurate for quantification of apoptotic cells. Unlike many other biomarkers of apoptotic cells, FLICAs can be used to detect apoptosis ex vivo and in vivo in different tissues.

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Extensive DNA fragmentation that generates a multitude of DNA double-stand breaks (DSBs) is a hallmark of apoptosis. We developed several variants of the widely used TUNEL methodology that is based on the use of exogenous terminal deoxynucleotidyl transferase (TdT) to label 3'OH ends in DSBs with fluorochromes. Flow- or image-cytometry is then employed to detect and quantify apoptotic cells labeled this way. Here, we describe a variant of this technique using BrdUTP as a TdT substrate. The incorporated BrdU is subsequently visualized by a fluorochrome-tagged antibody. This is a particularly simple, rapid, and sensitive approach to detect DSBs.We also describe modifications of the labeling protocol permitting the use of deoxyribonucleotides other than BrdUTP to label DSBs. Concurrent differential staining of cellular DNA and multiparameter analysis of cells by flow- or image-cytometry enable correlations between apoptosis induction and the cell cycle phase. Examples of the detection of apoptotic cells in cultures of human leukemic cell lines treated with TNF-α and DNA topoisomerase I inhibitor topotecan are presented. The protocol can be applied to cells treated with cytotoxic drugs in vitro, ex vivo, or to clinical samples.

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Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

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The predominant characteristic of malignant glioma is the presence of invading tumor cells in the peritumoral zone. Distinguishing between tumor cells and normal cells in a peritumoral lesion is challenging. Therefore, the aim of the present study was to investigate the cell-cycle phase measurements of fixed paraffin-embedded specimens from the peritumoral invading zone of high-grade gliomas using laser scanning cytometry. A total of 12 high-grade gliomas (2 anaplastic astrocytomas and 10 glioblastomas) were studied. The tumor core and peritumoral invading zone of each tumor specimen were investigated. Tissue sections (50 µm) from the paraffin blocks were deparaffinized, rehydrated and enzymatically disintegrated, and the cells in suspension were stained with propidium iodide and placed on microscope slides. A slight trend for an increased S-phase fraction in the peritumoral invading zone compared with the tumor core was observed (P=0.24). Additionally, there was a trend for a decrease in the overall survival time of patients with increasing peritumoral invading zone S-phase fraction (P=0.12). These data suggest that laser scanning cytometry is a powerful and clinically relevant tool for the objective analysis of the cell cycle in malignant gliomas.

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For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.

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During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

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The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.

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Since uterine cervical cancer is regarded as a radiosensive tumor, ionizing radiation is the most frequently used treatment modality against the disease. Although the crucial end-point is radiation-induced cell death, the tumors are not equally sensitive to radiation. Determining the criteria that may be used to predict tumor radiosensitivity is of importance; however, little success has been achieved thus far. In radioresistant cases the therapeutic strategy should be changed, thereby avoiding ineffective or unnecessary treatment. Furthermore, identification of the underlying molecular processes leading to radioresistance may lead to novel radiosensitising strategies. Cervical smears were obtained from seven patients with locally advanced cervical cancer following each radiotherapy, and the radiation-induced damage of cancer tissue was examined by routine cytology. Since the formation of DNA double-strand breaks is considered critical for the cytocidal effect of radiation therapy, the molecular changes of the neoplastic cells were also assessed by laser scanning cytometry (LSC). Radiation-induced morphological changes of cancer cells were evident at a dose of 7.2 Gy, whereas increased DNA content (or DNA index) was observed prior to the onset of morphological changes. Molecular change was detected earlier than the morphological change of the irradiated cancer cells, indicating the feasibility of LSC in predicting the radiosensitivity of cervical cancer tissue.

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Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

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We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4(Cdt2) which regulates the licensing factor Cdt1 and p21(WAF1) during the G1 to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21(WAF1), detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2'-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G2 phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21(WAF1) and Cdt1 was seen at the end of G1 phase and all DNA replicating cells were p21(WAF1) and Cdt1 negative. The loss of p21(WAF1) preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).

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The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

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Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA-associated DAPI fluorescence (represented by maximal pixels). This change is paralleled by an increase in nuclear area. Thus, the ratio of mean intensity of maximal pixels to nuclear area provides a very sensitive morphometric biomarker for the degree of senescence. This analysis is combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21(WAF1) ) and phosphorylation of ribosomal protein S6 (rpS6), a key marker associated with aging/senescence that is detected using a phospho-specific antibody. These biomarker indices are presented in quantitative terms defined as a senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. This system can be used to evaluate the anti-aging potential of test agents by assessing attenuation of maximal senescence. As an example, the inclusion of berberine, a natural alkaloid with reported anti-aging properties and a long history of use in traditional Chinese medicine, is shown to markedly attenuate the Mxt-induced SI and phosphorylation of rpS6. The multivariate analysis of senescence markers by laser scanning cytometry offers a promising tool to explore the potential anti-aging properties of a variety agents.

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Previous studies have shown that mild cognitive impairment (MCI) may be reflective of the early stages of more pronounced neurodegenerative disorders such as Alzheimer's disease (AD). There is a need for a minimally invasive and inexpensive diagnostic to identify those who exhibit cellular pathology indicative of MCI and AD risk so that they can be prioritized for primary preventative measures. The hypothesis was that a minimally invasive approach using cytological markers in isolated buccal mucosa cells can be used to identify individuals of both MCI and AD. An automated buccal cell assay was developed using laser scanning cytometry (LSC) to measure buccal cell type ratios, nuclear DNA content and shape, and neutral lipid content of buccal cells from clinically diagnosed AD (n = 13) and MCI (n = 13) patients prior to treatment compared to age- and gender-matched controls (n = 26). DNA content was significantly higher in all cell types in both MCI (P < 0.01) and AD (P < 0.05) compared with controls mainly due to an increase in >2N nuclei. Abnormal nuclear shape (circularity) was significantly increased in transitional cells in MCI (P < 0.001) and AD (P < 0.01) when compared to controls. In contrast, neutral lipid content (as measured by Oil red O "ORO" staining) of buccal cells was significantly lower in the MCI group (P < 0.05) compared with the control group. The ratio of DNA content/ORO in buccal basal cells for both MCI and AD was significantly higher compared to the control group, with ratios for MCI being approximately 2.8-fold greater (P < 0.01) and AD approximately 2.3-fold greater (P < 0.05) than the control group. Furthermore, there was a strong negative correlation between buccal cell DNA content and ORO content in the AD group (r(2) = 0.75, P < 0.0001) but not in MCI or controls. The changes in the buccal cell cytome observed in this study could prove useful as potential biomarkers in identifying individuals with an increased risk of developing MCI and eventually AD.

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Because it is not known exactly when or where myeloid dendritic cells (mDCs) acquire their atopic dermatitis (AD)-specific T-cell-polarising ability in patients with this condition, we used laser scanning cytometry (LSC) to determine whether isolated peripheral blood mDCs from AD patients differed from cells from controls in their cytokine expression profiles de novo and after stimulation with Staphylococcus enterotoxin B (SEB) and thymic stromal lymphopoietin (TSLP), which represents an AD-like microenvironment. Unstimulated mDCs from AD patients showed pluripotent T-cell-polarising capacity, and the surrounding skin microenvironment was essential for the distinctive, disease-specific activity of mDCs (Th2-Th22 bias). We also emphasise that LSC is an attractive technique to study the effect of new DC-targeted therapeutic modalities in AD.

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The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

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Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. © 2013 International Society for Advancement of Cytometry.

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Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.

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