Solid tumor preparation for clinical application of flow cytometry

Intense interest in advanced squamous cell cancers of the head and neck (SCC of H&N) has resulted from the recent progress made in tumor responses with chemotherapy and radiotherapy. Unfortunately, the response patterns and clinical outcome of such patients are not adequately predicted on an individual patient basis using clinical parameters or conventional morphology. The study of flow cytometrically determined cellular parameters in such tumors is therefore of interest, but is hindered by inadequate tumor preparative technology. The previous article (10) in this journal describes the use of a murine SCC tumor, LC12, which was employed for comparative testing and determination of optimum techniques of preparation for this tumor. This report describes the application of these techniques to 144 specimens of human SCC of H&N. The mean total yield for these specimens is 7.4 X 10(7) cells/g of tissue. The mean viable enzymatic yield (3.3 X 10(7) cells/g) was higher than the mean viable mechanical yield (2.0 X 10(7) cells/g) except when lymph nodes were the source of the specimen (5.4 X 10(7) cells/g). The mean dye exclusion viability from enzymatically dissociated specimens were above 90%. Significant aneuploidal subpopulation losses were evident in mechanically dissociated and enucleated specimens. 65% of the enzymatically dissociated specimens were successfully cultured with a mean cloning efficiency of 2.1 X 10(-3). Preparative techniques derived from comparative testing with a murine standard tumor have been successfully applied to 144 specimens of SCC of H&N with resultant high yields and excellent viability. Technical problems detected during the preliminary testing with LC12 were confirmed in the human tumors.     

Introduction to flow cytometry data file standard

The Data File Standards Committee of the Society for Analytical Cytology presents a Standard to be used for the storage of data associated with flow cytometric measurements. The Standard specifies a format that provides for the inclusion of all information necessary to fully describe: 1) the instrument used for the measurement; 2) the sample measured; 3) the data obtained; and 4) the results of analysis of the data. The Committee and the Society for Analytical Cytology point out that the use of this Standard by all those individuals and companies that generate or use data taken with flow cytometers or generate methods of analysis for the data will encourage the sharing of such data and methods of analysis.     

A reliable preparation of mono-dispersed chromosome suspensions for flow cytometry

Summary A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4° C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

Identifiability of a stochastic model for cell debris in flow cytometry

 One of the most important problems in recovering DNA distribution from flow cytometric DNA measurements is the presence of background noise. In this paper, we analyse a probabilistic model recently proposed for background debris distribution and based on a specific probabilistic mechanism for the DNA fragmentation process of the cell nucleus. In particular, we carry out some sufficient conditions to uniquely identify the original DNA distribution from the flow cytometric data. Received: 15 June 1997 / Revised version: 18 November 1997     

Isolation of murine microglial cells for RNA analysis or flow cytometry

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.     

Response of a brachytherapy model using 125I in a murine tumor system

The effects of low-dose-rate irradiation (brachytherapy) were investigated in vivo using a murine mammary adenocarcinoma (MTG-B) growing in the flank of C3H mice. For local tumor irradiations, a noninvasive cap was devised to cover the tumor and house three 125I seeds (average apparent activity 5.2 mCi each) located at 120 degree intervals around the circumference of the hemispherical cap (13 mm i.d.). Mice were secured during treatment in a tube allowing limited mobility while restricting access to the seeds. Tumors were exposed to a series of dose rates ranging from 14-40 cGy/h, and the total dose over the treatment interval (48 or 72 h) ranged from 830 to 2378 cGy. A total of nine experiments were conducted using the caps over a 10-week interval. In each experiment three groups (irradiated tumors, sham controls, and untreated controls) were analyzed, each containing 8-15 mice (N = 34, untreated control; N = 46, sham control; N = 91, brachytherapy irradiation). The brachytherapy results are compared to the effects of external beam irradiation in the same tumor system. A linear relationship was observed between the total radiation dose and doubling volume growth delay (GDDV) or treatment volume growth delay (GDTV) for the brachytherapy and external beam irradiation. The slopes of the dose-response curves are steeper for the acute dose (517 cGy/min) external beam irradiation (0.0072 day/cGy, GDDV; 0.00695 day/cGy, GDTV) than for the brachytherapy (0.0050 day/cGy, GDDV; 0.0057 day/cGy, GDTV) using both GDTV and GDDV end points. Comparison of the tumor volume regrowth slopes indicates that the tumor bed effect is larger for external beam irradiation than for brachytherapy, suggesting that the tumor bed effect may be dose-rate dependent.     

Enrichment for submitotic cell populations using flow cytometry

BACKGROUND: One of the most dramatic events during the course of the mammalian cell cycle is mitosis, when chromosomes condense and segregate, the nuclear envelope breaks down, and the cell divides into two daughter cells. Although cells undergoing mitosis are cytologically distinguishable from nonmitotic cells, few molecular markers are available to specifically identify mitotic cells, especially cells within different stages of mitosis. METHODS: We applied the flow cytometric method of Juan et al. (Cytometry 32:71-77, 1998) to obtain cells with various levels of the molecular markers cyclin B1 and phosphorylated histone H3; fluorescence microscopy was then used to identify sorted cells in different stages of mitosis. RESULTS: We observed the substantial enrichment of submitotic cell populations. CONCLUSIONS: This method represents an effective approach to obtain an enriched population of submitotic cells without the use of drug treatments or prior synchronization.     

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.     

Nonlinear studies of tumor morphological stability using a two-fluid flow model

Journal of Mathematical Biology - We consider the nonlinear dynamics of an avascular tumor at the tissue scale using a two-fluid flow Stokes model, where the viscosity of the tumor and host...     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

Abstract. The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Micro-MRI at 11.7 T of a murine brain tumor model using delayed contrast enhancement

In vivo imaging methodologies allow for serial measurement of tumor size, circumventing the need for sacrificing mice at given time points. In orthotopically transplanted murine models of brain tumors, cross-section micro-MRI allows for visualization and measurement of the physically inaccessible tumors. To allow for long resident times of a contrast agent in the tumor, intraperitoneal administration was used as a route of injection for contrast-enhanced micro-MRI, and a simple method for relative tumor volume measurements was examined. A strategy for visualizing the variability of the delayed tumor enhancement was developed. These strategies were applied to monitor the growth of brain tumors xenotransplanted into nude mice and either treated with the antiangiogenic peptide EMD 121974 or an inactive control peptide. Each mouse was used as its own control. Serial imaging was done weekly, beginning at Day 7 after tumor cell implantation and continued for 7 weeks. Images obtained were reconstructed on the MRI instrument. The image files were transferred off line to be postprocessed to assess tumor growth (volume) and variability in enhancement (three-dimensional [3-D] intensity models). In a small study, tumor growth and response to treatment were followed using this methodology and the high-resolution images displayed in 3-D allowed for straightforward qualitative assessment of variable enhancement related to vascular factors and tumor age.     

FuGEFlow: data model and markup language for flow cytometry

Background  

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata.