Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications.

Distinct effects of anti-tumor necrosis factor combined therapy on TH1/TH2 balance in rheumatoid arthritis patients.

The immune balance in patients with rheumatoid arthritis (RA), a disease characterized by TH1 dominance, treated by the preferred combined anti-tumor necrosis factor (anti-TNF) and methotrexate (MTX) therapy was evaluated by assessing the chemokine and cytokine receptors as well as apoptosis induction. A meta-analysis of combined therapy by TNF blockers and MTX in 15 RA patients, MTX monotherapy in 20 RA patients, and 11 diagnosed but untreated RA patients was performed by assessing several immune markers in the whole lymphocyte population, as well as in specific CD4 cells, by both flow cytometry and image analysis. A significant downregulation of CXCR3 and IL-12 receptors (both TH1 markers) and a significant increase in the chemokine receptor CCR4 and, to a lesser extent, IL-4R (both TH2 markers) were found; a particularly marked increase was found in patients treated by combined therapy. This phenomenon was pronounced in CD4 cells and was accompanied by a high proportion of apoptotic cells. The therapeutic effect of MTX and TNF blockers may be due to apoptosis induction in lymphocytes infiltrating from the inflammation site and restoring the TH1/TH2 balance.

An information-theoretical model for breast cancer detection.

OBJECTIVES: Formal diagnostic modeling is an important line of modern biological and medical research. The construction of a formal diagnostic model consists of two stages: first, the estimation of correlation between model parameters and the disease under consideration; and second, the construction of a diagnostic decision rule using these correlation estimates. A serious drawback of current diagnostic models is the absence of a unified mathematical methodological approach to implementing these two stages. The absence of a unified approach makes the theoretical/biomedical substantiation of diagnostic rules difficult and reduces the efficacy of actual diagnostic model application. METHODS: The present study constructs a formal model for breast cancer detection. The diagnostic model is based on information theory. Normalized mutual information is chosen as the measure of relevance between parameters and the patterns studied. The "nearest neighbor" rule is utilized for diagnosis, while the distance between elements is the weighted Hamming distance. The model concomitantly employs cellular fluorescence polarization as the quantitative input parameter and cell receptor expression as qualitative parameters. RESULTS: Twenty-four healthy individuals and 34 patients (not including the subjects analyzed for the model construction) were tested by the model. Twenty-three healthy subjects and 34 patients were correctly diagnosed. CONCLUSIONS: The proposed diagnostic model is an open one, i.e. it can accommodate new additional parameters, which may increase its effectiveness.

Methotrexate selectively modulates TH1/TH2 balance in active rheumatoid arthritis patients.

OBJECTIVE: The mechanism by which low dose methotrexate (MTX, the gold standard treatment for rheumatoid arthritis) exerts its anti-inflammatory effect in rheumatoid arthritis (RA) patients is still debated. Lately, the MTX immunosuppressive effect has been related to apoptosis, especially in active RA patients, with ROS involvement. METHODS: In the present research we investigated MTX oxidative effect and its ability to modulate immune balance in active versus non-active RA patients. RESULTS: Our results show that MTX induces IL-10 secretion (a TH2 cytokine) and significantly reduces TH1 profile in Peripheral Mononuclear Cells (PMNC) derived from active RA patients (n=28). Additionally, we found that MTX modulates the immune status towards TH2 dominance by decreasing the IL-12R and the CXCR3 receptors typical for the TH1 population. Moreover, MTX was found to inhibit the production of nitric oxide (NO) in these patients, a phenomenon that might contribute to MTX action toward cytokine homeostasis. A significant correlation was found between MTX IL-10 induction and NO inhibition in active RA patients. CONCLUSION: Our data suggest that, in active RA patients, apoptosis induction by MTX may be primarily due to IL-10 production via modulation of oxidative stress, which may restore the critically important immune balance. These findings may contribute to determining which group of RA patients may better respond to MTX therapy.

Low dose methotrexate induces apoptosis with reactive oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic lines.

BACKGROUND: The mechanism by which low dose methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effect in rheumatoid arthritis (RA) patients is still debated. Recently it has been related to the induction of apoptosis. OBJECTIVE: We investigated the degree of apoptotic induction by MTX in lymphocytic (Jurkat T, EL4 T, and Raji B) and monocytic cell lines (U937 and THP1) and its relation to reactive oxygen species (ROS) generation, as a possible mechanism underlying the apoptotic events. METHODS: All cell types were incubated with a range of MTX concentrations (0.001, 0.01, 0.1, 1, and 10 muM) for up to 24 h. Cytotoxicity was assessed by Trypan Blue exclusion and MTT test; cell size and granularity by forward and side scatters (FSC, SSC). Apoptosis was measured by Annexin V test and FDA polarization; and mitochondrial ROS generation by DHR123 probe and by NAC inhibition. RESULTS: MTX significantly reduced cell viability and proliferation in all cell lines, being most effective in the Jurkat T lymphocytic line. The MTX cytotoxicity (at the optimal concentrations corresponding to low dose MTX therapy) was attributed to apoptosis, as suggested by morphological changes (shrinkage, increased granularity) and confirmed by Annexin V binding and FDA hyperpolarization. The apoptotic induction and the ROS generation (statistically correlated to apoptosis) were most pronounced in the Jurkat and EL4 T cell lines, and were partially inhibited by the antioxidant N-acetyl L-cysteine (NAC). CONCLUSION: According to the present observations, MTX may most likely induce apoptosis through oxidative stress. The high susceptibility of T cell lines to MTX induced apoptosis may account for the beneficial effect of MTX treatment in rheumatoid arthritis, which is characterized by hyperproliferation of T cells.

The influence of different cultivating conditions on polymorphonuclear leukocyte apoptotic process in vitro, II: ultrastructural characteristics of PMN populations incubated with proteinase 3 anti-neutrophil autoantibodies.

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27: 23-32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40min and 12h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named "first," "second," and "third." The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the "first" line is characterized by intensification and modification of activation; the "second" by vacuolized cell forms; and the "third" by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.

Overexpression of 15-lipoxygenase in the vascular endothelium is associated with increased thymic apoptosis in LDL receptor-deficient mice.

BACKGROUND: 15-Lipoxygenase (15-LO) is a nonheme iron-containing enzyme that catalyzes the peroxidation of fatty acids. Herein, we studied the effect of 15-LO overexpression in the vascular endothelium on thymocyte apoptosis by evaluating thymuses from low-density lipoprotein receptor-deficient (LDL-RD) mice and LDL-RD/15-LO mice. Thymuses were evaluated by immunohistochemistry and by TUNEL whereas in vitro studies were carried out by employing freshly isolated thymocytes from the respective mice and evaluation of apoptosis by propidium iodide and annexin V cytometry. METHODS AND RESULTS: The apoptotic index in LDL-RD/15-LO mice was significantly higher than in the LDL-RD mice. In the thymic medulla the difference was smaller, although still significant. Freshly isolated thymus cells from LDL-RD/15-LO mice exhibited a higher rate of spontaneous cell death than controls. Incubation of thymus cells in the presence of the cell-permeable caspase-3 inhibitor DEVD-CMK resulted in a decrease in the frequency of apoptotic cells in LDL-RD/15-LO thymocytes, whereas no effect was evident in control thymocytes. The antioxidant N-acetylcysteine causes the increase in apoptosis in both groups. CONCLUSION: LDL-RD/15-LO mice exhibit increased thymocyte apoptosis both in vivo and in vitro. These findings may suggest a role for 15-LO in the natural selection of thymocytes.

Antineutrophil cytoplasmic autoantibodies penetrate into human polymorphonuclear leukocytes and modify their apoptosis.

OBJECTIVE: The interaction of extracellular anti-neutrophil cytoplasmic autoantibodies (ANCA) with neutrophilic granules may play an important role in the pathogenesis of ANCA-related disorders. It has been confirmed that apoptosis is an essential trigger associated with translocation of the cytoplasmic granules to the cell surface, and with the expression of ANCA antigens. Since cell penetration by autoantibodies and apoptosis may be associated processes, we tested the hypothesis that penetration of ANCA-autoantibodies into polymorphonuclear leukocytes (PMNs) has an effect on apoptosis and thereby can influence surface antigen expression. METHODS: PMNs were isolated from the blood of healthy volunteers and incubated in the presence of anti-proteinase3 (PR3) enriched IgG or normal human IgG. For each period of incubation (40 minutes or 12 hours) we evaluated: 1) PMN morphology by light microscopy (LM) and transmission electron microscopy (TEM) for general estimation of the apoptotic process, and 2) ANCA binding to the target antigen by immunogold electron microscopy (IgEM). RESULTS: Both normal and anti-PR3 IgG penetrate PMNs. The labeled PR3-ANCA were localized on PR3 granules, regardless of the granules' location within the cell, and in the sites where the PMN destruction processes were most expressed. The destructive processes showed extensive apoptotic characteristics, in contrast to PMNs penetrated by normal IgG. CONCLUSION: PR3 ANCA penetrate PMNs and, via the interaction between PR3-ANCA and PR3-containing granule components, initiate a modification of the apoptotic process.

The influence of different cultivating conditions on polymorphonuclear leukocyte apoptotic processes in vitro, I: the morphological characteristics of PMN spontaneous apoptosis.

Polymorphonuclear leukocyte (PMN) populations incubated in vitro with normal human serum are save-regulated systems of spontaneous apoptosis. Light microscopy (LM), transmission (TEM), and scanning (SEM) electron microscopes were used for the evalution of PMN apoptopic alteration. Twelve-hour PMN populations were represented by optimal number of normal and different apoptotic forms. Their ultrastructural analysis showed that on this background, 3 apoptotic cell lines (code named "first," "second," and "third") were predominated. The following characteristics were featured: "first"--vacuolization of same organelles, release of their content outside, increase of general cytoplasmic density, nuclear filling with condensed chromatin, and formation of PMNs mainly into small, round, dense forms; "second"--involvement of micronuclei or nuclei in apoptosis, their displacement to the cytoplasmic membrane and separation from the cells, and cytoplasm had numerous intact granules almost until the completion of apoptosis; "third"--synchronous apoptotic process of the nuclei and cytoplasm, moderate electronic density of cytoplasm, and granular translocation to the cell surface. Secondary necrosis was completed mainly in the apoptotic process of the "second" and "third" lines. SEM surfaces confirmed the results of TEM. This research showed that neutrophil spontaneous apoptosis is a complicated process. The 3 apoptotic cell lines reflect different pathways characteristic for the studied systems under certain conditions of cultivation.

Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements.

Apoptosis is a dynamic process of variable duration. The ability to continuously detect the death process occurring in single or subgroups of cells is therefore very important in identifying apoptotic cells within a complex population. The Individual Cell Scanner (ICS), a multiparametric, multilaser-based scanning static cytometer, was used in the present report for the continuous monitoring of the apoptosis process. Fluorescence intensity (FI), polarization (FP), kinetic measurements, and cluster analysis of subpopulations were carried out utilizing various fluorescent probes. Hydrogen peroxide-induced apoptosis was monitored online in intact live lymphocytes by continuous sequential measurements of intracellular hyperpolarization. Plasma membrane asymmetry, mitochondrial membrane potential, and lysosomal rupture were monitored in individual cells. Cytoplasmic condensations, due to cell shrinkage and early lysosomal rupture, were found to be very early events of apoptosis. The new analytical capabilities suggested here may provide simple and convenient methods for detecting apoptosis from its earlier stages.

Analysis of laser scattering pattern as an early measure of apoptosis.

Light scattering pattern analysis (LSPA) was applied in the current study for accurate and sensitive detection of subtle changes in cell size, which occur in mouse thymocytes undergoing apoptosis. The decrease in cell diameter as measured by LSPA was found to be an early signal of apoptosis preceding the externalization of phosphatidylserine on the outer membrane. When apoptosis was induced by dexamethasone, the change in cell size was dose and time dependent, and could be blocked by pretreatment of the thymocytes with N-acetylcysteine (NAC). This implies that the scattering pattern, when combined with fluorescent markers such as annexine-V, may be a powerful tool for early detection of apoptosis. Another advantage gained by the use of this method is the ability to repeatedly trace the same cells and to monitor the kinetics of their size changes.

Fluorescein fluorescence hyperpolarization as an early kinetic measure of the apoptotic process.

The ability to identify apoptotic cells within a complex population is crucial in the research and diagnosis of normal physiology and disease states. The Cellscan mark S (CS-S) cytometer was used in this study to detect intracellular fluorescence intensity and polarization (FI and FP) in several well-established models of apoptosis: Following spontaneous apoptosis, as well as glucocorticoid or anti Fas-induced apoptosis, CS-S individual cell-based analysis revealed the appearance of a cell cluster characterized by low FI and high FP. Temporal analysis of annexine V binding and FP measurements following DXM treatment showed that hyperpolarization preceded phosphatidylserine appearance on the outer plasma membrane. The early increase in FP was found to be dose dependent and inversely related to cell diameter. Cell dehydration and alteration of plasma membrane transport properties, both occurring during early stages of apoptosis, may be involved in the phenomena of intracellular fluorescein hyper-polarization in apoptosis.

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements.

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.

Determination of cellular thiol levels in individual viable lymphocytes by means of fluorescence intensity and polarization.

Cellular thiol levels regulate lymphocyte proliferation and death and play a significant role in the immune response. Therefore, the ability to analyze the total protein and non-protein thiol compounds and their distribution among individual living lymphocytes is of great importance. A quantitative measurement of intracellular sulphydryl groups in living lymphocytes using the Cellscan mark F (CS-F) cytometer, in conjunction with the probe CMFDA, is described. This technique permits the detection, identification, and study of sub-populations and single cells in a sample of heterogeneous lymphocytes. The Cellscan apparatus is a laser based scanning cytometer incorporating a unique cell carrier which allows repeated, high-precision measurements of fluorescence intensity (FI) and fluorescence polarization (FP) to be made on intact individual living cells under controlled physiological conditions. The discernible effect of fluorophore molecules bound to thiols having a higher FP than free molecules was used to estimate their relative fractions in living lymphocytes. The results were more conspicuous when the ratio between FP measured at two wavelengths (FPR) of the fluorogenic molecules was used for analysis. In addition, the intracellular dynamic changes in the FI, FP and FPR of the fluorescent probe were also monitored. The cellular sulphydryl content of each lymphocyte within a population was recorded by the CS-F, and sub-populations or individual cells were classified according to their thiol levels and their metabolic rates. Changes in thiol concentration were observed following mitogenic activation of peripheral lymphocytes.

Reactivity of peripheral blood lymphocytes to oxidized low-density lipoprotein: a novel system to estimate atherosclerosis employing the Cellscan.

BACKGROUND: The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population. HYPOTHESIS: In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls. METHODS: The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases. RESULTS: Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 micrograms/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 micrograms/ml). Maximal depolarization was shown upon triggering with 2 micrograms/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 +/- 0.05 and 1.028 +/- 0.055, respectively). CONCLUSION: These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.

The trace and subgrouping of lymphocyte activation by dynamic fluorescence intensity and polarization measurements.

Cell activation involves conformational changes of cytosolic enzymes, and/or their regulatory proteins, as well as intracellular matrix re-organization. In this work, these changes were monitored by dynamic measurements of fluorescence polarization in single cells incubated with or without phytohaemagglutinin (PHA), using the Cellscan mark S (CS-S) cytometer. This instrument and the procedure used proved to be a powerful tool for distinguishing subpopulations of cells. Grouping of cells by their staining rates (the time rate of change of the fluorescence intensity) yielded three major subgroups. For each subgroup, the fluorescence depolarization (FDP) induced by the incubation with PHA was measured. The kinetics of the subgroups indicate that the major FDP is contributed by the cells with the lowest staining rate. This FDP is approximately 1.5 times greater than that of a bulk population. It is believed that the analysis of kinetic probing might yield an important and more sensitive method for functional marking of subgroups of cells by their response characteristics.

Indication that intracellular fluorescence polarization of T lymphocytes is cell cycle dependent.

The degree of depolarization of fluorescence light emitted from an organic dye, used as a molecular probe, is a powerful tool in probing the microenvironment. Polarization measurements of intracellular exogenous fluorescein have been shown to reflect the physiological state of the cells. The relationship between intracellular fluorescein fluorescence polarization (IFFP) and cell cycle, was investigated in the leukemia T-lymphocyte Jurkat cell line. Jurkat T cells were cultured in increasing cell densities, their cell cycle progression cytometrically monitored and the IFFP measured. At the highest cell density, the subpopulation of cells at the resting phases the (Gzero/G1) predominated, and the mean IFFP was 0.186 +/- 0.015. At the lowest density, with diminished proportion of cells in the G1/G2 stages the mean IFFP decreased to 0.126 +/- 0.01. Treatment of the Jurkat T cell line with phase arrested agents 1 microM hydroxyurea, or 1 microM nocodazole, arrests the cells in the S and G2/M phases, respectively. These treated cells exhibit significantly lower IFFP values, mean polarization value 0.140, as compared to 0.171 +/- 0.009 in control cells. Preincubation of Jurkat cells in buffer in accumulation of the cells in the Gzero/G1 phases as well as a parallel increase in IFFP. A characteristic decrease in IFFP was demonstrated upon triggering these cells with Phytohaemagglutinin (PHA). High correlation (Pearson correlation = 0.942) was found between percentage of cells in the Gzero/G1 phases and the mean IFFP of the measured cell population. These results may indicate that the intracellular microviscosity of Jurkat T cells as measured by IFFP, is changing over the cell cycle.