scholarly journals Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.

1978 ◽  
Vol 26 (11) ◽  
pp. 921-933 ◽  
Author(s):  
J Fried ◽  
A G Perez ◽  
B D Clarkson
Keyword(s):  
S Phase ◽  
Staining Procedure ◽  
Flow Cytofluorometry ◽  
Monolayer Cultures ◽  
Nonionic Detergent ◽  
Hela S3 ◽  
Hela S3 Cells ◽  
Dna Histograms ◽  
Nonionic Detergents

The rapid hypotonic staining procedure developed by Krishan for DNA determinations by flow cytofluorometry has been proven accurate for in vivo cell samples and for cell lines growing in suspension culture. We show that the unmodified procedure may produce distorted DNA histograms when used for staining cells growing in monolayer cultures, however. To eliminate these distortions, it was necessary to avoid the use of trypsin by staining the attached cells directly, using a hypotonic fluorochrome solution to which nonionic detergent was added. Two sublines of HeLa S3 cells are shown to exhibit major differences in their staining characteristics. By using our revised staining procedure, the two sublines appear to produce very satisfactory DNA histograms. However, in only one subline does the S phase fraction calculated from the histograms agree with the autoradiographical labeling index. Mitotic cells remain intact under these staining conditions, and the principal observed effect of nonionic detergents in this case is to decrease the coefficient of variation of fluorescence intensity.

scholarly journals Oncolytic Vaccinia Virus Harboring Aphrocallistes vastus Lectin Inhibits the Growth of Cervical Cancer Cells Hela S3

Marine Drugs ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. 532
Author(s):  
Jiajun Ni ◽  
Hualin Feng ◽  
Xiang Xu ◽  
Tingting Liu ◽  
Ting Ye ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Vaccinia Virus ◽  
Cancer Cells ◽  
Inhibitory Effect ◽  
Genes Encoding ◽  
Cervical Cancer Cells ◽  
Hela S3 ◽  
Hela S3 Cells

Aphrocallistes vastus lectin (AVL) is a C-type marine lectin produced by sponges. Our previous study demonstrated that genes encoding AVL enhanced the cytotoxic effect of oncolytic vaccinia virus (oncoVV) in a variety of cancer cells. In this study, the inhibitory effect of oncoVV-AVL on Hela S3 cervical cancer cells, a cell line with spheroidizing ability, was explored. The results showed that oncoVV-AVL could inhibit Hela S3 cells growth both in vivo and in vitro. Further investigation revealed that AVL increased the virus replication, promote the expression of OASL protein and stimulated the activation of Raf in Hela S3 cells. This study may provide insight into a novel way for the utilization of lection AVL.

scholarly journals Probability paper analysis of flow cytofluorometric measurements of cellular deoxyribonucleic acid content.

1981 ◽  
Vol 29 (7) ◽  
pp. 851-857 ◽  
Author(s):  
J E Neely ◽  
J W Combs
Keyword(s):  
Acid Content ◽  
Deoxyribonucleic Acid ◽  
Paper Analysis ◽  
S Phase ◽  
Flow Cytofluorometry ◽  
Normal Distributions ◽  
Probability Paper ◽  
Graphic Analysis ◽  
Dna Contents ◽  
Dna Histograms

A graphic analysis of cellular deoxyribonucleic acid (DNA) histograms obtained by flow cytofluorometry is described. This technique utilizes probability paper that graphs data in cumulative percentile form. From this graphic representation, individual normal distributions contributing to the histogram may be isolated and statistically describe. G0G1 and G2M distributions are independently described and S phase is fit to 1--5 overlapping normal distributions as dictated by the analysis results. This technique offers several advantages, including relatively simple mathematical calculations and few assumptions or restraints placed on the analysis. It has proved to be useful in analyzing proliferating and nonproliferating populations as well as distributions with unusual findings such as debris or aberrant DNA contents.

scholarly journals A rapid single step staining technique for DNA analysis by flow microfluorimetry.

1980 ◽  
Vol 28 (9) ◽  
pp. 1021-1024 ◽  
Author(s):  
I W Taylor
Keyword(s):  
Dna Analysis ◽  
Staining Technique ◽  
Single Step ◽  
Cell Nuclei ◽  
Hoechst 33342 ◽  
Monolayer Cultures ◽  
Nonionic Detergent ◽  
Staining Techniques ◽  
Triton X ◽  
Dna Histograms

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.

In Vitro Culture of Human Thyroid Cells: Preliminary Findings on the Role of the Pathological Condition of the Donors

1997 ◽  
Vol 25 (2) ◽  
pp. 153-160
Author(s):  
Francesca Mattioli ◽  
Marianna Angiola ◽  
Laura Fazzuoli ◽  
Francesco Razzetta ◽  
Antonietta Martelli
Keyword(s):  
Pathological Condition ◽  
Primary Cultures ◽  
S Phase ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Toxicological Studies ◽  
Predictive Indicator

Although primary cultures of human thyroid cells are used for endocrinological and toxicological studies, until now no attention has been paid toward verifying whether the hormonal conditions to which the gland was exposed in vivo prior to surgery could influence in vitro responses. Our findings suggest that the hormonal situation in vivo cannot be used as a predictive indicator of triiodothyronine and thyroxine release and/or S-phase frequency in vitro, either with or without the addition of bovine thyrotropin.

scholarly journals Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Robert J. Francis ◽  
Gillian Robb ◽  
Lee McCann ◽  
Bhagwati Khatri ◽  
James Keeble ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Large Field ◽  
Staining Procedure ◽  
3D Analysis ◽  
Mycobacterium Tuberculosis Infection ◽  
In Vivo Models ◽  
Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

scholarly journals PDTM-17. MiR-126, miR-369-5p AND miR-487b DRIVE PEDIATRIC GLIOBLASTOMA INVASION VIA KCNA1

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi190-vi191
Author(s):  
Yulun Huang ◽  
Lin Qi ◽  
Mari Kogiso ◽  
Yuchen Du ◽  
Frank Braun ◽  
...  
Keyword(s):  
Cell Migration ◽  
Neurological Deficits ◽  
Integrated Analysis ◽  
Normal Brain ◽  
Tumor Cell Migration ◽  
Monolayer Cultures ◽  
Invasive Capacity ◽  
Resected Primary Tumor

Abstract Diffuse invasion is one of the key features that make GBM particularly difficult to treat. We hypothesize that direct comparison of matched invasive (GBMINV) and tumor core GBM cells (GBMTC) would facilitate the discovery of drivers of pediatric GBM (pGBM) invasion. However, GBMINV cells are extremely difficult to obtain from normal brain tissues because aggressive surgical resection of normal tissue carries the risk of serious neurological deficits. Most past and current studies on GBM invasion were and are forced to utilize the resected primary tumor masses. To overcome this barrier, we utilized a panel of 6 pediatric patient tumor-derived orthotopic xenograft (PDOX) mouse models to isolate matching pairs of GBMTC cells and GBMINV cells and confirmed a significantly elevated invasive capacity in GBMINV cells both in vitro and in vivo. Global profiling of 768 human microRNA using a real-time PCR-based Taqman system identified 23 microRNAs were upregulated in the GBMINV cells in at least 4 of the 6 pGBM models as compared with the matching GBMTC cells. We subsequently showed that silencing the top three miRNAINV, miR-126, miR-369-5p, and miR-487b, suppressed tumor cell migration in vitro (both as neurospheres and monolayer cultures) without affecting cell proliferation, and blocked pGBM invasion in mouse brains. Integrated analysis of the mRNA profiling of the same set of GBMTC and GBMINV cells revealed the affected signaling pathways and identified KCNA1 as the sole common computational target gene of the three miRNAINV. Treatment of three pairs of GBMTC and GBMINV cells with two KCNA1 inhibitors, ADWX1 and Agitoxin 2, caused significant suppression of pGBM cell migration in vitro. In conclusion, this study revealed an intrinsically elevated invasive phenotype in GBMINV cells, identified miR-126, -369-5p, and -487b as novel drivers of pGBM invasion, and characterized KCNA1 as a potential therapeutic target for arresting pGBM invasion.

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