Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry

2016 ◽  
Vol 2016 (10) ◽  
pp. pdb.prot087247 ◽  
Author(s):  
Lisa C. Crowley ◽  
Grace Chojnowski ◽  
Nigel J. Waterhouse
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Propidium Iodide Staining

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1558-1558 ◽  
Author(s):  
Maria Bhatti ◽  
Thomas Ippolito ◽  
Cory Mavis ◽  
Matthew J. Barth
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Mtor Pathway ◽  
Propidium Iodide Staining ◽  
Induction Of Apoptosis ◽  
Pre Treatment

Abstract Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL. Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays. Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites. Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. Disclosures No relevant conflicts of interest to declare.

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

scholarly journals Evolution of Genome Size in Duckweeds (Lemnaceae)

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Wenqin Wang ◽  
Randall A. Kerstetter ◽  
Todd P. Michael
Keyword(s):  
Flow Cytometry ◽  
Genome Size ◽  
Propidium Iodide ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Continuous Increase ◽  
Spirodela Polyrhiza ◽  
Wolffia Arrhiza ◽  
Basic Reference

To extensively estimate the DNA content and to provide a basic reference for duckweed genome sequence research, the nuclear DNA content for 115 different accessions of 23 duckweed species was measured by flow cytometry (FCM) stained with propidium iodide as DNA stain. The 1C-value of DNA content in duckweed family varied nearly thirteen-fold, ranging from 150 megabases (Mbp) in Spirodela polyrhiza to 1,881 Mbp in Wolffia arrhiza. There is a continuous increase of DNA content in Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia that parallels a morphological reduction in size. There is a significant intraspecific variation in the genus Lemna. However, no such variation was found in other studied species with multiple accessions of genera Spirodela, Landoltia, Wolffiella, and Wolffia.

scholarly journals ONC-212 (I-39), a Novel Inhibitor of the UPR, Is Cytotoxic and Cytostatic Against CLL Cells Under in Vitro Conditions That Mimic the Tumor Microenvironment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3145-3145
Author(s):  
Narjis Rizwan ◽  
Yandong Shen ◽  
Edwin Iwanowicz ◽  
Stephen P. Mulligan ◽  
Kyle R Crassini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Effective Treatment ◽  
Cell Line ◽  
Propidium Iodide ◽  
Cycle Arrest ◽  
Migratory Capacity ◽  
Ic50 Values ◽  
Cells Cultured

Abstract Introduction Despite the revolution in the treatment of chronic lymphocytic leukemia (CLL) over the past decade with the introduction of novel inhibitors targeting the B-cell receptor (BCR) signaling pathway and the Bcl-2 family of proteins, relapse is still common. Recent studies suggest that imipridones, a novel class of small molecule agents that attenuate mitochondrial respiration and modulate an immune response against cancer cells, may be an effective treatment option for several difficult to treat cancers. We investigated the effects of the imipridone, ONC-212 (I-39, first published by Nanjing Gator Meditech), as a potential therapeutic strategy for CLL using the OSU-CLL cell line and a modified OSU-CLL line in which TP53 was stably knocked out and primary CLL cells cultured under conditions that mimic the tumour microenvironment (TME). Methodology Primary CLL cells were co-cultured with CD40L-expressing fibroblasts to mimic aspects of the TME. The cytotoxicity of ONC-212 was assessed using the mitochondrial dye DiIC1(5), propidium iodide and flow cytometry. The effects of the drug on the adhesive and migratory capacity of primary CLL cells were evaluated using antibodies against CD49d, CXCR4 and an in vitro migration assay using stroma-derived factor 1a (SDF1-α). Changes in protein expression were assessed by immuno-blotting. The effects of ONC-212 on the cell cycle and proliferation were assessed using the OSU-CLL cell line. OSU-CLL cells were modified using the CRISPr-Cas9 technology to be TP53 deficient (OSU-TP53ko). The proportion of cells in each cycle phase was determined using propidium iodide and flow cytometry. Cell proliferation rates were determined using carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. Results ONC-212 induced apoptosis in a dose-dependent manner in primary CLL cells cultured in medium alone or in contact with CD40L-fibroblasts (Figure 1); the IC50 values were 72.97 nm +/- 1.45 nM and 472 +/- 2.04 nM, respectively. OSU-CLL and OSU-TP53ko cells were also sensitive to ONC-212, although the TP53 deficient line was less sensitive than OSU-CLL(Figure 1). IC50 values for the cell lines were 22 +/- 1.37 nM (OSU-CLL) and 48 +/- 3.25 nM (OSU-TP53ko). ONC-212 induced cell cycle arrest of the OSU-CLL and OSU-TP53ko lines at the G1/S phase transition. This effect was concomitant with a significant reduction in the proliferation of both lines. ONC-212 significantly down-regulated expression of the adhesion molecule CD49d and the G-coupled protein receptor CXCR4 on primary CLL cells. Down-regulation of CXCR4 translated into a decrease in the migratory capacity of CLL cells along an SDF1-α gradient. Immunoblotting suggested the mechanisms of action of ONC-212 include inhibition of ERK1/2-MAPK, a decrease in the Bcl-2/Bax ratio and upregulation of the pro-apoptotic Puma and Bak proteins. Conclusions ONC-212 is highly effective against CLL cells at nanomolar concentrations, against cells cultured under conditions that mimic aspects of the TME and against TP53-deficient cells. ONC-212 has cytotoxic effects, induces cell cycle arrest, slows proliferation and inhibits the mechanisms by which CLL cells migrate to and are retained within the TME. ONC-212 inhibited signaling downstream of the BCR and induced a pro-apoptotic 'tipping' of the balance in expression of BCl-2 family proteins. These data suggest ONC-212 may represent an effective treatment for CLL, particularly for patients who have high risk, relapsed/refractory disease associated with loss or mutation of TP53. Disclosures No relevant conflicts of interest to declare.

Urine cell flow cytometry analysis of PD-L1 expression and DNA content for bladder cancer.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 466-466
Author(s):  
Pin-I Chen ◽  
Alice (Xiaoyang) Wang ◽  
Mustafa Deebajah ◽  
Shaheen Alanee ◽  
Bruce Kendrick Patterson
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Bladder Cancer ◽  
Epithelial Cells ◽  
Cancer Patient ◽  
Cancer Patients ◽  
Dna Content ◽  
Urine Samples ◽  
Normal Donor ◽  
Bladder Cancer Patient

466 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method to unbiasedly analyze PD-L1 expression and DNA content in cells from urine samples will help us better understand bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in urine samples were pelleted, fixed/permeabilized (in incellMAX, IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression. The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However, increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. In addition, a comparison of urine collection fixatives showed that incellMAX-fixed samples had the best single cell recovery and DNA content measurement, as shown by lower cell cycle dye staining variability (lower rCV). Statistically significant differences were found between cancer patients and normal samples. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples. Comparing urine samples from bladder cancer patients and normal yielded statistically significant differences that could provide valuable information for bladder cancer patient management.

Abstract 218: Nuclear Remodeling Following Mechanical Circulatory Support

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Jun Luo ◽  
Stephen Farris ◽  
Deri Helterline ◽  
April Stempien-Otero
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Content ◽  
Left Ventricular ◽  
Ventricular Assist Devices ◽  
Circulatory Support ◽  
Ventricular Assist ◽  
Imaging Flow Cytometry ◽  
Nuclear Number ◽  
Nuclear Remodeling

Rationale: Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates the analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox. Objective: We used a novel technique-imaging flow cytometry-to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart. Methods and Results: Cardiomyocytes were isolated from 8 subjects undergoing primary heart transplantation and 15 subjects following unloading with left ventricular assist device (LVAD, mean unloading time 13.7 ± 9.1 months). Myocyte size, nuclear number and size, DNA content (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Myocyte size and nuclear morphology was not significantly different between the groups. However, DNA content per nucleus was significantly decreased (P < 0.01) and the correlation between nuclear size and DNA content lost. The frequency of the cell cycle markers, Ki67 and phospho-histone3 (H3P) were not increased after hemodynamic unloading. Conclusions: Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.

scholarly journals Preliminary Characterization of a Monoclonal Antibody (AS-2) against Cell Cycle Related Proteins

1998 ◽  
Vol 17 (2) ◽  
pp. 93-101
Author(s):  
Stefano Nigro ◽  
Anna Rapallo ◽  
Angela Di Vinci ◽  
Elio Geido ◽  
Roberto Orecchia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Dna Content ◽  
Staining Pattern ◽  
Isolated Nuclei ◽  
Erythroleukemia Cell ◽  
Related Proteins

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described.AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

scholarly journals Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

2006 ◽  
Vol 188 (16) ◽  
pp. 5958-5965 ◽  
Author(s):  
Samer Sakr ◽  
Melilotus Thyssen ◽  
Michel Denis ◽  
Cheng-Cai Zhang
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Division ◽  
Cell Growth ◽  
Cell Size ◽  
Dna Content ◽  
Small Cells ◽  
Filamentous Cyanobacterium ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N2. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.

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