Immunofluorescence analysis of cytokeratin 8/18 staining is a sensitive assay for the detection of cell apoptosis

Dong, Qiao‐Mei; Ling, Chun; Zhao, Li

doi:10.3892/ol.2015.2856

Cited by 11 publications

(14 citation statements)

References 23 publications

(22 reference statements)

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“…Apoptosis-related proteins can be localized and quantified by immunofluorescence. 32 , 33 Cleaved caspase-3 has been used as a marker for cell apoptosis. Increased expression of the proapoptotic protein Cleaved caspase-3 means apoptosis of tumor cells.…”

Section: Resultsmentioning

confidence: 99%

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells

Li¹,

Yin²,

Ji³

et al. 2018

DDDT

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BackgroundTo improve the targeting ability of antitumor drugs, we identified the antigens with high expression on the surface of tumor cells associated with tumor escape, such as the complement regulatory protein CD55 molecule, which is also known as the decay accelerating factor. In this study, phage display technology was used to screen and identify CD55-specific ligand peptide (CD55sp) bound to CD55 molecule on the surface of cervical cancer HeLa cells. We then explored the role of this peptide in inhibiting the growth of cervical cancer cells in vitro. Our characterization of CD55sp will provide implication for tumor target therapy.MethodsThe phage bound to the surface of HeLa cells were isolated by phage display technology. Positive phage clones were identified by ELISA. Phage was then amplified and determined by agarose gel electrophoresis after monoclonal DNA extraction. DNA sequencing and bioinformatical analysis were conducted to obtain specific ligand peptides. Flow cytometry and immunofluorescence were used to measure the expression of CD55 molecule on the surface of tumor and normal cells. Subsequently, the effects of CD55sp on the proliferation and apoptosis of HeLa and SiHa cells were determined by Cell Counting Kit-8 (CCK-8), flow cytometry, and TUNEL assay, respectively. The morphology of apoptotic cells was examined by electron microscope. The distribution of Cleaved caspase-3 was detected by immunofluorescence. The expression of bcl-2 and Cleaved caspase-3 were determined by Western blot.ResultsThe results showed that the peptide (QVNGLGERSQQM) can bind to the CD55 molecule on the surface of cervical cancer HeLa and SiHa cells as a ligand peptide. It can also effectively inhibit the proliferation of cervical cancer cells and induce cell apoptosis.ConclusionThis study demonstrates that CD55sp screened by phage display technology plays a strong antitumor role.

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Section: Resultsmentioning

confidence: 99%

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells

Li¹,

Yin²,

Ji³

et al. 2018

DDDT

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“…It is noteworthy to mention that in case of the CellSearch isolated CTCs, when ferrofluid was not covering the cell surface, some granular structures could be observed on the CTC surfaces, as indicated by arrows in Figure 4 and Figure S1 . A speckled CK pattern in the respective immunofluorescence images would confirm the reorganization of their cytoskeleton and their early stage of apoptosis [ 17 , 30 , 31 ], whereas a fragmented nucleus would even indicate advanced apoptosis [ 30 ]. However, in many CTCs where the punctuated CK is clearly distinguishable, the cells are fully covered by the ferrofluid, hindering the observation of blebs on the cells.…”

Section: Discussionmentioning

confidence: 99%

Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles

Nanou

Flohr

Bono

et al. 2018

Cancers

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To explore morphological features of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs), we developed a protocol for scanning electron microscopy (SEM) of CTCs and tdEVs. CTCs and tdEVs were isolated by immunomagnetic enrichment based on their Epithelial Cell Adhesion Molecule (EpCAM) expression or by physical separation through 5 μm microsieves from 7.5 mL of blood from Castration-Resistant Prostate Cancer (CRPC) patients. Protocols were optimized using blood samples of healthy donors spiked with PC3 and LNCaP cell lines. CTCs and tdEVs were identified among the enriched cells by fluorescence microscopy. The positions of DNA+, CK+, CD45− CTCs and DNA−, CK+, CD45− tdEVs on the CellSearch cartridges and microsieves were recorded. After gradual dehydration and chemical drying, the regions of interest were imaged by SEM. CellSearch CTCs retained their morphology revealing various shapes, some of which were clearly associated with CTCs undergoing apoptosis. The ferrofluid was clearly distinguishable, shielding major portions of all isolated objects. CTCs and leukocytes on microsieves were clearly visible, but revealed physical damage attributed to the physical forces that cells exhibit while entering one or multiple pores. tdEVs could not be identified on the microsieves as they passed through the pores. Insights on the underlying mechanism of each isolation technique could be obtained. Complete detailed morphological characteristics of CTCs are, however, masked by both techniques.

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“…The nuclei were stained using DAPI (4′, 6-diamidino-2-phenylindol). Here, we monitored the existence of Dil + /cytokeratin-19 + cells (dual red/green stained cells), indicating the differentiation of transplanted cells into the pneumocyte-like cells [35].…”

Section: Immuno Uorescence Imaging (If)mentioning

confidence: 99%

C-Kit+ Cells Can Modulate Asthmatic Condition via Differentiation Into Pneumocyte-Like Cells and Alteration of Inflammatory Responses via ERK/NF-κB Pathway

Mirershadi

Ahmadi

Rahbarghazi‬

et al. 2021

Preprint

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Background The exact role of the progenitor cell types in the dynamic healing of asthmatic lungs is lacking. This investigation was proposed to evaluate the effect of intra-tracheally administered rat bone marrow-derived c-kit⁺ cells on ovalbumin-induced sensitized male rats. Methods Forty rats were randomly divided into 4 groups; healthy rats received phosphate-buffered saline (PBS) (C); sensitized rats received PBS (S); PBS containing C-kitˉ cells (S + C-kit−); and PBS containing C-kit⁺ cells (S + C-kit⁺). After two weeks, circulatory CD4⁺/CD8⁺ T-cell counts and pulmonary ERK/NF-κB signaling pathway as well as the probability of cellular differentiation were assessed. Results The results showed that transplanted C-Kit+ cells were engrafted into pulmonary tissue and differentiated into epithelial cells. C-Kit+ cells could increase the number of CD4+ cells in comparison with S group (p < 0.001); however, diminished the level of CD8+ cells (p < 0.01). Moreover, data showed increased p-ERK/ERK ratio (p < 0.001) and NF-ƙB level (p < 0.05) in sensitized rats compared to C group. The administration of C-kit+, but not C-Kit−, decreased p-ERK/ERK ratio and NF-ƙB level than those of S group (p < 0.05). Conclusions The study showed that C-Kit+ cells engrafted into pulmonary tissue reduced NF-ƙB protein level and diminished p-ERK/ERK ratio, leading to suppression of inflammatory response in asthmatic lungs.

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Immunofluorescence analysis of cytokeratin 8/18 staining is a sensitive assay for the detection of cell apoptosis

Cited by 11 publications

References 23 publications

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells

A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells

Scanning Electron Microscopy of Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles

C-Kit+ Cells Can Modulate Asthmatic Condition via Differentiation Into Pneumocyte-Like Cells and Alteration of Inflammatory Responses via ERK/NF-κB Pathway

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