Measurement Of DNA Content Research Articles

Comparative DNA Flow Cytometric Study of Primary Intraocular and Central Nervous System Lymphomas

Primary intraocular lymphoma is generally considered as a subset of primary CNS lymphoma. This study attempts to show that they may in fact represent distinct entities by comparing their respective proliferation rates using DNA flow cytometry. Four samples of primary intraocular lymphoma and seven samples of primary CNS lymphoma were analyzed, all from paraffin-embedded tissue. All tumors were of the large B-cell type. A normal human tonsil sample was used as a control. Tissue samples were analyzed by DNA flow cytometry, which is a precise and objective method to measure DNA content and cell proliferation of a tumor. S-phase fraction (SPF) and DNA content were measured for each sample. The average SPF for primary intraocular lymphoma was significantly higher than that of primary CNS lymphoma, 23.8 (range: 18.9 to 29.6) versus 15.1 (range: 1.1 to 25.1) respectively. Of the 11 tumors analyzed, 2 brain tumors were aneuploid and 1 eye tumor was peridiploid. All other tumors were diploid. Thus, no significant pattern was detected in the DNA content of the tumors. This lack of clinical significance of tumor aneuploidy is consistent with data reported in the literature. The results of this study indicate that primary intraocular lymphoma is more aggressive and of higher grade than primary CNS lymphoma. The different proliferation rates of intraocular and CNS lymphomas may be explained by either their different spatial location or a distinct genetic composition, the latter reinforcing the hypothesis that the two are fundamentally different entities

Simple Field Storage of Fish Samples for Measurement of DNA Content by Flow Cytometry.

Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species‐specific standard, after 1, 5, and 10 days storage at 0–4°C or freezing at −80°C. The fluorochrome 4′,6‐diamidine‐2′‐phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Nuclear DNA content as an indicator of inflorescence colour stability of in vitro propagated solid and chimera mutants of chrysanthemum

In chrysanthemum, breeders seek for desirable characteristics of the inflorescence, which can first be established once the plant is mature. The present study aims to determine whether measurement of DNA content can be useful in the detection of somaclonal variants and/or separation of chimera components in chrysanthemum at the early in vitro multiplication stage. Eleven Chrysanthemum × morifolium (Ramat.) Hemsl. cultivars of the Lady group (a mother cultivar and ten of its radiomutants obtained by X-ray- or γ-irradiation; solid and periclinal chimeras) were propagated in vitro. Single-node explants were cultured in Murashige and Skoog (MS) medium, either without plant growth regulators (PGRs) or supplemented with 6-benzyladenine (BA) and indole-3-acetic acid (IAA). The nuclear DNA content was measured by flow cytometry (FCM) in the shoots produced in vitro. After acclimatization and growth of the plants in a glasshouse, inflorescence colour was recorded. The addition of PGRs to the medium almost doubled the mean number of shoots produced in vitro per explant, but caused a change in inflorescence colour of all (‘Lady Apricot’; periclinal chimera) or part of the plants (‘Lady Amber’; solid mutant and ‘Lady Salmon’; periclinal chimera). All radiomutants contained less DNA than the mother cultivar ‘Richmond’. There were significant differences in DNA content between plants of the same cultivar grown in media with or without PGRs for ‘Lady Apricot’ and ‘Lady Salmon’, but no phenotype alternation occurred in chrysanthemums produced in PGR-free medium compared to the original cultivars. Conversely, in medium with PGRs, chimeras produced flowers different from the original colour. In all except one cultivar (‘Lady Amber’; solid mutant) a lack of differences in genome size between plants grown in either medium coincided with a stable inflorescence colour. The occurrence of some plants of ‘Lady Amber’ with different inflorescence colour may be due to small DNA changes, undetectable by FCM. It can be concluded that FCM analysis of DNA content in young plantlets can be indicative of the stability of inflorescence colour in chrysanthemum, especially chimeric cultivars, and for mutant detection.

Introduction to Multiparametric Flow Cytometry and Analysis of High-Dimensional Data.

Multiparametric flow cytometry is a technique utilized in translational experiments that utilizes fluorescently tagged antibodies and functionalfluorescent dyesto measure proteins on the surface or in the cytoplasm of cells and to measure processes occurring within cells themselves. These fluorescent molecules, or fluorophores, can be tagged to antibodies to measure specific biological molecules such as proteins inside or on the surface of cells. Small organic compounds such as the nucleic acid binding dye propidium iodide (PI) can permeate compromised cell membranes when cells are no longer viable or used to measure DNA content of cycling cells. Successful completion of flow cytometry experiments requires expertise in both the preparation of the samples, acquisition of the samples on instruments, and analyses of the results. This chapter describes the principles needed to conduct a successful multiparameter flow cytometry experiment needed for drug development with references to well established internet resources that are useful to those less experienced in the field. In addition, we provide a brief introduction to data analysis including complex analysis of 10+ parameters simultaneously. These high-dimensional datasets require novel methods for analysis due to the volume of data collected, which are also introduced in this chapter.

Abstract 4043: SYD985 synergy with PARP inhibitors (PARPi) in human tumor cell lines and patient-derived xenografts (PDXs)

Abstract SYD985 is a HER2-targeting antibody-drug conjugate comprised of a cleavable linker and the prodrug seco-DUBA which, upon release, spontaneously rearranges to form the active toxin DUBA. DUBA covalently binds adenine in the DNA resulting in DNA damage, cell cycle perturbation and associated cytotoxicity. SYD985 induces dose-dependent cytotoxicity of human tumor cell lines and PDXs expressing HER2 on the outer cell membrane. The cytotoxicity of PARPi depends on PARP trapping, i.e. the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of clear1 and unclear origins2,3. We investigated potential synergy in cytotoxic activity of SYD985 and the PARPi niraparib and olaparib towards HER2-expressing tumor cells in vitro and in vivo. Tumor cell lines were from breast (BT-474, AU-565, UACC-893) or ovarian (TOV-112D, NIH:OVCAR-3) origin. Cytotoxicity was established by measuring DNA content and metabolic activity of living cells. For both PARPi the highest non-cytotoxic concentration for each cell line was established. This concentration was used to measure the potential potency shift of the SYD985 concentration-response curve by comparing SYD985 IC50 values in the absence and presence of PARPi. PARPi-induced fold change in IC50 for SYD985 was established in two independent experiments (N=2, n=3). Niraparib, dosed at a non-cytotoxic concentration, induced a 1.5 to 136-fold potentiation of SYD985-induced tumor cell killing. In BT-474 cells, niraparib co-treatment unveils a biphasic concentration-response curve to SYD985. This is in line with the fact that BT-474 cells express high levels of HER2 and are known to respond to naked trastuzumab at concentrations slightly above those of SYD985. Olaparib, dosed at a non-cytotoxic concentration, induced a 0.4 to 7.5-fold potentiation of SYD985-induced tumor cell killing. In a breast cancer PDX mouse clinical trial in immunodeficient mice that lack carboxylesterase 1c (Ces1c) we studied the combination of SYD985 (1 mg/kg, IV, SD) and niraparib or olaparib (50 mg/kg Q1DX21, IP). In order to detect potential additive and/or synergistic effects of SYD985 and PARPi, a suboptimal dose of SYD985 (1 mg/kg) was selected. PARPi doses were chosen known to inhibit growth of tumors with a defective DNA repair mechanism4. Data clearly show that the combination of SYD985 and PARPi are more effective in tumor volume reduction than either monotherapy alone. In conclusion, niraparib and olaparib act in synergy with SYD985 in killing human HER2-expressing tumor cells in vitro, both in high HER2 (BT-474, AU-565, UACC-893) and low HER2 (NIH:OVCAR-3; TOV-112D) expressing cells. In vivo such synergistic activity appears to translate in improved tumor growth inhibition of various breast cancer PDX models. 1Zimmermann M, Nature 559, 2018; 2Pommier Y, Sci Transl Med 8, 2016; 3Lord C, Science 355, 2017; 4Henneman L, PNAS 112, 2015 Citation Format: Wim H. Dokter, Fred A. Dijcks, Patrick G. Groothuis, Ellen Mattaar, Eline Loosveld, Daniëlle Jacobs, Monique van der Vleuten, Jan H. Schellens, Miranda van der Lee. SYD985 synergy with PARP inhibitors (PARPi) in human tumor cell lines and patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4043.

Overlapping region reconstruction in nuclei image segmentation

Automatic screening systems play an increasingly important role in the diagnosis of pathologists. Image measurement and classification are the key techniques of automatic screening systems, which directly determine the performance. The distortion in grey and texture after overlapping nuclei segmentation seriously degrades the DNA content measurement and nuclei classification. In order to solve this problem, this paper presents a new method to reconstruct the pixels in overlapping regions based on the GMM-UBM (Gaussian mixture model–universal background model). In this method, a large amount of data are first used to train a GMM (named UBM). Then, the GMM of each nucleus is derived by maximizing a posteriori adaptation with the UBM and the normal grey value of this nucleus. The grey values are randomly generated by the GMM and filled to the overlapping region, with the offset to fine-tuning the Gaussian components. Finally, the image inpainting algorithm is used to repair the connected region. Experimental results show that this method can effectively recover the nucleus features, such as texture, grey and optical density, and improve the accuracy of nucleus measurement and classification.

Abstract 373: Impacts of Harvesting Sites on Cardiac Extracellular Matrix Hydrogels Properties

Introduction: Cardiac extracellular matrix (cECM) hydrogel has been explored to treat myocardial infarction (MI). It possesses organ specific microenvironmental cues for resident cells and can be minimally invasive delivered into the heart using a transendocardial catheter. Recent clinical studies demonstrated the safety and feasibility of using cECM for treating MI patients. However, little is known about whether the region of cardiac tissue harvesting is critical for downstream applications. We performed in vitro studies to compare cECM hydrogels derived from porcine whole heart (WH), left ventricle (LV), and right ventricle (RV). Materials and Methods: Adult porcine hearts were obtained from a local USDA approved abattoir. The tissues were separated into 3 major categories: LV, RV, and WH tissues. Following our published protocol, tissues were decellularized and solubilized. The complete decellularization was confirmed by H&E staining and DNA content measurement. Chemical composition was examined using SDS-PAGE electrophoresis as well as Collagen and GAGs measurements. The gelation time, SEM (hydrogel microstructures), and mechanical properties (storage and complex modulus) were studied. Human cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) were separately seeded on the hydrogels to examine their responses. Cell viability and vascular differentiation of human MSCs were examined. Results and Discussion: The cECM from WH has more complex chemical composition compared with cECM from LV and RV. All three types of cECM hydrogels share many similarities in terms of their microstructure, gelation time and mechanical properties. WH-derived cECM hydrogels have larger variations in storage modulus (G’) and complex modulus (G*) compared with the other two types of cECM hydrogels. Both human CMs and MSCs could maintain high cell viability on all hydrogels. Regardless of harvesting sites, all cECM hydrogels have equivalent potency in directing vascular differentiation of seeded human MSCs. Conclusions: The cECM hydrogels from WH, LV and RV exhibited similarity in material properties and cell response in vitro . Future fabrication of cECM hydrogels from WH would increase the yield and decrease production cost.

Phenotypic and molecular characterization of polyploidy Vicia faba induced by colchicine

To induce polyploidy in diploid control plant of Vicia faba, seeds were socked in various concentrations of colchicine (0.1, 0.2, 0.3 and 0.4 %) for 9 and18 h. Putative ploidy plants were confirmed by screening morphological characters, measuring DNA content via spectrophotometer and analyzing stomatal behavior. Polyploidy plants showed slower growth rate along with increased potency and thickened large leaves. Three types of polyploidy were achieved; triploid, tetraploid and mixoploid. In induced polyploid plants, the length and width of stomata were increased, while the index of stomata was declined, in comparison to the initial diploid plants. Treatment with 0.3 and 0.4 % colchicine for 9 and 18 h were efficiently induced polyploidy. The highest stomatal size (22.53 µm) and widest stomatal aperture (9.72 µm) and the lowest stomatal index (34.24) where obtained when 0.4 % of colchicine where applied at 18hr tested time with high significant differences at p<0.05 in comparison to dipliod plants. We recommend this method for induction of polyploids in other different crops and possibly will selected these tetraploid plants for further breeding purposes.

Derivatives of usnic acid cause cytostatic effect in Caco-2 cells

Usnic acid has anti-cancer activity, however, low solubility and toxicity limit the potential. To investigate biological activity of usnic acid derivatives, enantiopure derivatives were synthesised by reacting usnic acid with ethylenediamine, which yielded one dimer product ((+)-1), and two tetra cyclic compounds ((+)-2 and (-)-2). The products were characterised with NMR, and evaluated in vitro in human colon cancer cell line Caco-2 by cell count, phase-contrast microscopy, MTT-assay, measurement of DNA content and cell cycle distribution. All compounds tested showed cytostatic effect in Caco-2 cells, but each compound had a distinct cellular effect. Compound (+)-1 showed anti-proliferative activity by increasing the percentage of cells in S-phase with 25% compared to the control. Compounds (+)-2 and (-)-2 induced paraptosis, but only compound (+)-2 modulated cell cycle distribution by accumulating cells in G2/M-phase by 47% and reduced DNA content by 60%. All compounds express interesting cellular and potential anti-proliferative activity.

Abstract P3-06-07: ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer

Abstract Background: Adhesion G protein-coupled receptors (GPCRs) form the second largest GPCR subfamily and control many cellular processes involved in cancer. ADGRF1 is an adhesion GPCR overexpressed and gene amplified in HER2+ and triple-negative breast cancer (BC). We have previously found a role of ADGRF1 in promoting tumorigenicity and metastatic processes in cell-based studies in HER2+ BC. The goal of this study was to evaluate the effects of ADGRF1 overexpression in vivo in HER2+ BC. Methods: BT474 stable cell lines were generated using lentiviral system containing ADGRF1 cDNA using PHAGE system, overexpressing ADGRF1 in Doxycycline (Dox)-inducible manner. Mice (N=8/group) were injected with various numbers of cells with (+) or without (-) Dox (10K, 30K, 100K, 300K, 1 million) to assess for tumor initiation and growth. Transcriptomic and proteomic analysis of ADGRF1-overexpressing BT474 cells was also performed using RNAseq and RPPA analysis, respectively. The candidates were validated using western blotting and IHC staining. Cell cycle analysis was conducted by measuring DNA content with propidium iodide using flow cytometry. Results: Dox-induced overexpression of ADGRF1 in mice led to inhibition of tumor growth compared to no Dox treated tumors. Furthermore, fewer mice injected with Dox-treated cells formed tumors. Our RNA-Seq data revealed downregulation of genes involved in cell cycle regulation, which included E2F targets, G2M checkpoint pathways, and MYC targets. Likewise in RPPA analysis, several proteins in the cell cycle pathway (FOXO1, AuroraA, Ki67, Ezh2, CHAF1, DEPTOR, Rb1, Phospho-Rb (S780), and Hexokinase II) were downregulated by ADGRF1 overexpression. Validation of RNAseq and RPPA candidates demonstrated lower number of Ki67+ cells and reduced NF-kB expression, and inhibition of STAT3 phosphorylation. Furthermore, we found that ADGRF1 overexpression by Dox also led to cell cycle arrest at GO/G1 phase. Conclusion: Our results suggest that ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer. These results are in contrast to the in vitro data demonstrating a role of ADGRF1 in promoting tumorigenicity and metastatic processes in HER2+ BC. Understanding the mechanism of this difference is critical in investigating ADGRF1 as a drug target in breast cancer. Citation Format: Raksha Bhat, Noor Mazin Abdulkareem, Lanfang Qin, Carmine De Angelis, Hala Yasser, Hariprasad Thangavel, Jennifer Torres, Vidyalakshmi Sethunath, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Martin Shea, Rachel Schiff, Meghana Trivedi. ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-07.

Genome size and SCoT markers as tools for identification and genetic diversity assessment in Echinacea genus

The Echinacea (coneflower) genus includes herbaceous flowering species mainly used for medicinal purposes, as well as the ornamental plants. Morphological similarities between Echinacea species often lead to their incorrect identification and characterization. Genome size and SCoT markers were used for identification and establishing the taxonomic relationships within Echinacea genus. The analysis of the nuclear DNA content indicated, that the majority of Echinacea species possessed intermediate genomes, and only E. pallida has a large genome size. 2C DNA content ranged from 14.31 pg/2C (E. simulata) to 28.59 pg/2C (E. pallida). Based on flow cytometric measurements (FCM) of the nuclear DNA content it was possible to distinguish three coneflower species, E. simulata, E. pallida and E. sanguinea, confirming validity of the method for initial species identification. Whereas, SCoT-PCR analysis enabled the identification of all studied accessions. Nine primers revealed polymorphism both between species, as well as varieties of E. angustifolia and E. paradoxa, demonstrating the usefulness of SCoT primers for genetic diversity studies in this genus. The phylogenetic analysis confirmed, that Echinacea species are closely related and within the investigated accessions two main phylogenetic groups exist, with one species (E. purpurea) being not clustered to any of the groups. Two methods, FCM and SCoT-PCR, can be successfully applied in identification and taxonomic relationship assessment, as well as in determination of the genetic diversity of Echinacea genus. Moreover, this is the first report on genome size that includes eight Echinacea accessions.

Dispersion stability and biocompatibility of four ligand-exchanged NaYF4: Yb, Er upconversion nanoparticles

Surface modification to obtain high dispersion stability and biocompatibility is a key factor for bio-application of upconversion nanoparticles (UCNPs). A systematic study of UCNPs modified with four hydrophilic molecules separately, comparing their dispersion stability in biological buffers and cellular biocompatibility is reported here. The results show that carboxyl-functionalized UCNPs (modified by 3,4-dihydrocinnamic acid (DHCA) or poly(monoacryloxyethyl phosphate (MAEP)) with negative surface charge have superior even-distribution in biological buffers compared to amino-functionalized UCNPs (modified by (aminomethyl)phosphonic (AMPA) or (3-Aminopropyl)triethoxysilane (APTES)) with positive surface charge. Subsequent investigation of cellular interactions revealed high levels of non-targeted cellular uptake of the particles modified with either of the three small molecules (AMPA, APTES, DHCA) and high levels of cytotoxicity when used at high concentrations. The particles were seen to be trapped as particle-aggregates within the cellular cytoplasm, leading to reduced cell viability and cell proliferation, along with dysregulation of the cell cycle as assessed by DNA content measurements. The dramatically reduced proportion of cells in G1 phase and the slightly increased proportion in G2 phase indicates inhibition of M phase, and the appearance of sub-G1 phase reflects cell necrosis. In contrast, MAEP-modified UCNPs are bio-friendly with increased dispersion stability in biological buffers, are non-cytotoxic, with negligible levels of non-specific cellular uptake and no effect on the cell cycle at both low and high concentrations. MAEP-modified UCNPs were further functionalized with streptavidin for intracellular microtubule imaging, and showed clear cytoskeletal structures via their upconversion luminescence. Statement of significanceUpconversion nanoparticles (UCNP) are an exciting potential nanomaterial for bio-applications. Their anti-Stokes luminescence makes them especially attractive to be used as imaging probes and thermal therapeutic reagents. Surface modification is the key to achieving stable and compatible hydrophilic-UCNPs. However, the lack of criteria to assess molecular ligands used for ligand exchange of nanoparticles has hampered the development of surface modification, and further limits UCNP’s bio-application. Herein, we report a systematic comparative study of modified-UCNPs with four distinct hydrophilic molecules, assessing each particles’ colloidal stability in biological buffers and their cellular biocompatibility. The protocol established here can serve as a potential guide for the surface modification of UCNPs in bio-applications.

Estimation of chromosome number among the progeny of a self-pollinated population of triploid Senno (Lychnis senno Siebold et Zucc.) by flow cytometry

Flow cytometry is a powerful tool to determine ploidy level and is commonly applied to measure DNA content and analyze the cell cycle. Here, we analyzed ploidy among the progeny derived from a population of self-pollinated triploid Senno (Lychnis senno Siebold et Zucc.). Each plant within the progeny was estimated to be an aneuploid showing a different ploidy level. For a large population, identification of chromosome number by chromosome observation is time-consuming. Therefore, a simple procedure to categorize different ploidy levels and chromosome number is required to facilitate breeding efforts. We applied flow cytometry to estimate chromosome number using internal standards for reference; these estimates were compared with the results obtained following chromosome observation. Leaf samples from progeny of triploid Senno were measured by flow cytometry in combination with an internal standard to evaluate DNA content accurately. By using appropriate internal standards, chromosome number was effectively estimated. When the parent triploid plant (strain MS, 2n = 3x = 36) was used as the internal standard, diploid (2n = 24) and different aneuploids (2n = 25, 44, 45, 55) were discriminated. For measurement of presumed triploids, strain No. 27 (2n = 25) was used as another internal standard. As a result, triploid (2n = 3x = 36) and different aneuploids (2n = 33, 35) were detected. The exact number of chromosomes as per chromosome observation corresponded well with the number estimated through flow cytometry. This procedure will facilitate ready, accurate, and reliable estimation of chromosome number using an adequate reference sample for internal standard. We discuss the possibility to estimate chromosome number by flow cytometry and the applicability of the procedure for determining the ploidy level as well as aneuploids in large populations derived from parental triploid Senno plants.

Comparison of Proliferation and Differentiation of Human Osteoblast-like Cells Harvested During Implant Osteotomy Preparation Using Two Different Drilling Protocols.

Autogenous bone grafts are considered a "gold standard." The success of autografts mainly depends on their ability to promote an osteogenic response. The aim of this study was to collect autogenous bone during implant osteotomy preparation using two different drilling protocols and to evaluate and compare the proliferation and differentiation ability of the collected bone particles. Autogenous bone particles were harvested from 20 patients during implant osteotomy preparation using two different drilling protocols: (1) standard drilling protocol with saline irrigation (according to the manufacturer's recommendation) and (2) low-speed drilling protocol without saline irrigation (speed < 200 rpm). Bone samples collected were cultured in growth medium, and after 2 to 3 weeks, cells that grew out from bone grafts were cultured in the normal medium as well as in osteogenic medium for days 0, 4, 7, and 20. Scanning electron microscopy, alizarin red/toluidine blue staining, DNA, ALP, and calcium content measurements were performed. Repeated measures analysis of variance (ANOVA) with Bonferroni's test was employed to analyze the data of this study. The total DNA content was significantly higher for the low-speed drilling samples compared with the standard drilling on day 4 (P < .05), day 7 (P < .01), and day 20 (P < .001) in the normal medium and on day 7 (P < .01) and day 20 (P < .01) in the osteogenic medium. Besides, calcium measurements and mineralized matrix formation observed with alizarin red/toluidine blue staining were significantly higher for the low-speed drilling group compared with the standard drilling group. Osteogenic efficacy (differentiation and proliferation) of autogenous bone particles collected using low-speed drilling was superior compared with standard drilling samples.

Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage.

This unit describes immunocytochemical detection of histone H2AX phosphorylated on Ser-139 (γH2AX) to reveal DNA damage, particularly when the damage involves the presence of DNA double-strand breaks (DSBs). These breaks often result from DNA damage induced by ionizing radiation or by treatment with anticancer drugs such as DNA topoisomerase inhibitors. Furthermore, DSBs are generated in the course of DNA fragmentation during apoptosis. The unit presents strategies to distinguish radiation- or drug-induced DNA breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes immunocytochemical detection of γH2AX combined with measurement of DNA content to identify cells that have DNA damage and concurrently to assess their cell-cycle phase. The detection is based on indirect immunofluorescence using FITC- or Alexa Fluor 488-labeled antibody, with DNA counterstained with propidium iodide and cellular RNA removed with RNase A. © 2019 by John Wiley & Sons, Inc.

Induction of Polyploidy by Colchicine on the Green Algae Dunaliella salina

In this study, induction of polyploidy by colchicine was investigated in Dunaliella salina. To induce polyploidy in the cells, different concentrations of colchicine (0.05, 0.1, 0.5 and 1%) were used in three periods of times 12, 24, and 36 h. Measurement of DNA content was done by Nano drop method. All concentrations of colchicine caused an increase in DNA content. Results showed that the highest value of DNA obtained related to algal cells were exposed to 1.0 and 0.5% colchicine for 36 h (0.213 μg/μL and 0.274 μg/μL, respectively), with respect to DNA content in control sample, these treatments caused induction of polyploidy in algal cells. Survival percentage of cells at all concentrations of colchicine significantly decreased. Based on obtained results from survival rate and DNA content, two concentrations of 0.1 and 0.5% colchicine were selected for other analysis. Colchicine treatments (0.1 and 0.5%) caused an increase in doubling time and specific growth rate declined in the treated algae. Pictures obtained by fluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear size of the microalgae. Results of flow cytometry analysis indicated that percentage of polyploid cells in cultures treated by 0.1% colchicine was 58.26% and by 0.5% colchicine was 74.19%.

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

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 ( &gt; 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.

Tunable Properties of Poly-DL-Lactide-Monomethoxypolyethylene Glycol Porous Microparticles for Sustained Release of Polyethylenimine-DNA Polyplexes.

Direct pulmonary delivery is a promising step in developing effective gene therapies for respiratory disease. Gene therapies can be used to treat the root cause of diseases, rather than just the symptoms. However, developing effective therapies that do not cause toxicity and that successfully reach the target site at therapeutic levels is challenging. We have developed a polymer-DNA complex utilizing polyethylene imine (PEI) and DNA, which was then encapsulated into poly(lactic acid)-co-monomethoxy poly(ethylene glycol) (PLA-mPEG) microparticles via double emulsion, solvent evaporation. Then, the resultant particle size, porosity, and encapsulation efficiency were measured as a function of altering preparation parameters. Microsphere formation was confirmed from scanning electron micrographs and the aerodynamic particle diameter was measured using an aerodynamic particle sizer. Several formulations produced particles with aerodynamic diameters in the 0-5μm range despite having larger particle diameters which is indicative of porous particles. Furthermore, these aerodynamic diameters correspond to high deposition within the airways when inhaled and the measured DNA content indicated high encapsulation efficiency. Thus, this formulation provides promise for developing inhalable gene therapies.

Measuring DNA content in live cells by fluorescence microscopy

BackgroundLive-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.ResultsPresented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.ConclusionsThis method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.