Assessment of oxidative metabolism

Oxidative metabolism is one of the central physiological processes that regulate multiple functions in a cell including cell death and survival, proliferation, gene transcription, and protein modification. There are multitudes of techniques that are used to evaluate oxidative activity. Here, we summarize how to measure oxidative activity by flow cytometry. This versatile technique allows the evaluation of the level of oxidative activity within heterogeneous populations of cells and in cell culture. Flow cytometry is a quick method that yields highly reproducible results with small sample volumes. Therefore, it is an ideal technique for evaluating changes in oxidative activity in samples from mice

Flow-Cytometric Phosphoprotein Analysis Reveals Agonist and Temporal Differences in Responses of Murine Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells (HSCs) are probably the best-studied adult tissue-restricted stem cells. Although methods for flow cytometric detection of phosphoproteins in hematopoeitic progenitors and mature cells are available, analogous protocols for HSC are lacking. We present a robust method to study intracellular signaling in immunophenotypically-defined murine HSC/progenitor cell (HPC)-enriched populations. Using this method, we uncover differences in the response dynamics of several phosphoproteins representative of the Ras/MAP-Kinase(K), PI3K, mTOR and Jak/STAT pathways in HSC/HPCs stimulated by Scf, Thpo, as well as several other important HSC/HPC agonists

Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFκB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFα, and interferon), and we demonstrate scalability by printing a chip with ∼4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability

Multiparameter Phospho-Flow Analysis of Lymphocytes in Early Rheumatoid Arthritis: Implications for Diagnosis and Monitoring Drug Therapy

The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality.PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed.Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness

Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.</p> <p>Results</p> <p>Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC₅₀for myosin light chain phosphorylation using Y27632 was determined to be 2.1 ± 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods.</p> <p>Conclusion</p> <p>Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.</p

Specific cellular signal-transduction responses to in vivo combination therapy with ATRA, valproic acid and theophylline in acute myeloid leukemia

Acute myeloid leukemia (AML) frequently comprises mutations in genes that cause perturbation in intracellular signaling pathways, thereby altering normal responses to growth factors and cytokines. Such oncogenic cellular signal transduction may be therapeutic if targeted directly or through epigenetic regulation. We treated 24 selected elderly AML patients with all-trans retinoic acid for 2 days before adding theophylline and the histone deacetylase inhibitor valproic acid (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22), and sampled 11 patients for peripheral blood at day 0, 2 and 7 for single-cell analysis of basal level and signal-transduction responses to relevant myeloid growth factors (granulocyte-colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, interleukin-3, Flt3L, stem cell factor, erythropoietin, CXCL-12) on 10 signaling molecules (CREB, STAT1/3/5, p38, Erk1/2, Akt, c-Cbl, ZAP70/Syk and rpS6). Pretreatment analysis by unsupervised clustering and principal component analysis divided the patients into three distinguishable signaling clusters (non-potentiated, potentiated basal and potentiated signaling). Signal-transduction pathways were modulated during therapy and patients moved between the clusters. Patients with multiple leukemic clones demonstrated distinct stimulation responses and therapy-induced modulation. Individual signaling profiles together with clinical and hematological information may be used to early identify AML patients in whom epigenetic and signal-transduction targeted therapy is beneficial

A Quantitative Image Cytometry Technique for Time Series or Population Analyses of Signaling Networks

Background: Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. Methodology/Principal Findings: We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. Conclusions/Significance: The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging

Supermultiplexed optical imaging and barcoding with engineered polyynes

Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening

FoxM1, a Forkhead Transcription Factor Is a Master Cell Cycle Regulator for Mouse Mature T Cells but Not Double Positive Thymocytes

FoxM1 is a forkhead box transcription factor and a known master regulator required for different phases of the cell cycle. In cell lines, FoxM1 deficient cells exhibit delayed S phase entry, aneuploidy, polyploidy and can't complete mitosis. In vivo, FoxM1 is expressed mostly in proliferating cells but is surprisingly also found in non-proliferating CD4+CD8+ double positive thymocytes. Here, we addressed the role of FoxM1 in T cell development by generating and analyzing two different lines of T-cell specific FoxM1 deficient mice. As expected, FoxM1 is required for proliferation of early thymocytes and activated mature T cells. Defective expression of many cell cycle proteins was detected, including cyclin A, cyclin B1, cdc2, cdk2, p27 and the Rb family members p107 and p130 but surprisingly not survivin. Unexpectedly, loss of FoxM1 only affects a few cell cycle proteins in CD4+CD8+ thymocytes and has little effect on their sensitivity to apoptosis and the subsequent steps of T cell differentiation. Thus, regulation of cell cycle genes by FoxM1 is stage- and context-dependent