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Nanomechanical Analysis of Cells from Cancer Patients
Published in Lajos P. Balogh, Nano-Enabled Medical Applications, 2020
Sarah E. Cross, Yu-Sheng Jin, Jianyu Rao, James K. Gimzewski
Two types of triple labelling assays were performed: One was DNA/F-actin/Ber-EP4 and the other DNA/Calretinin/Ber-EP4. For both experiments, cells were fixed first with 3.7% formaldehyde for 30 min at room temperature and then washed with 1 PBS three times, then incubated with 1% BSA in PBS pH 7.4 × for 30 min. For DNA/F-actin/Ber-EP4 labelling, cells were first incubated with mouse anti-human Ber-EP4 (DAKO) at 1:300 dilution for 1 h, followed by Cy3-conjugated AffiniPure goat anti-mouse IgG(H + L) (Jackson ImmunoResearch Lab) at 1:200 dilution for 30 min, then with BODIPY FL phallacidin F-actin (Molecular Probes) at 1:40 dilution for 30 min. Finally, cells were incubated with 1:10,000 DAPI for 5 min. For DNA/Calretinin/Ber-EP4 labelling, cells were first incubated with mouse anti-human Ber-EP4 (DAKO) at 1:300 dilution for 1 h, followed by Cy3-conjugated AffiniPure goat anti-mouse IgG(H L) (Jackson ImmunoResearch Lab) at 1:200 dilution for 30 min. + Cells were then further incubated with 1:600 diluted rabbit anti-human Calretinin antibody (Zymed) for 1 h then with FITC-conjugated AffiniPure goat anti-rabbit IgG(H + L) (Jackson ImmunoResearch Lab) at 1:50 dilution for 30 min, followed by 1:10,000 DAPI for 5 min. All incubations were performed at room temperature and there were three PBS washing steps in between. Cells were covered with mounting medium for fluorescence microscopic examination. Images were taken using an Olympus BX-40 microscope with a × 40 objective.
A Strategy for Regeneration of Three-Dimensional (3D) Microtissues in Microcapsules: Aerosol Atomization Technique
Published in Naznin Sultana, Sanchita Bandyopadhyay-Ghosh, Chin Fhong Soon, Tissue Engineering Strategies for Organ Regeneration, 2020
Chin Fhong Soon, Wai Yean Leong, Kian Sek Tee, Mohd Khairul Ahmad, Nafarizal Nayan
DAPI (4’, 6-diamidino-2-phenylindole dihydrochloride) staining was performed to investigate the cells or nuclei distribution in the microtissues after reaching dormant phase. For staining agent preparation, DAPI (0.1 μg/ml) (Sigma Aldrich, St. Louis, MO, USA) was diluted in HBSS. Then, the microcapsules of HaCaT were washed in HBSS and then incubated in DAPI solution for 20 min in the dark. Subsequently, the DAPI stain was removed and the stained microtissues were washed with HBSS solution. The stained microtissues images were viewed and captured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) mounted with a DP73 CCD camera (Olympus, Tokyo, Japan).
Structure-Function Elucidation of Flavonoids by Modern Technologies
Published in Dilip Ghosh, Pulok K. Mukherjee, Natural Medicines, 2019
Ritu Varshney, Neeladrisingha Das, Rutusmita Mishra, Partha Roy
DAPI or 4′,6-diamidino-2-phenylindole is a florescent dye used to stain the nucleus of a cell. This stain has the property of binding to the A-T rich regions in the DNA (Eriksson et al. 1993) and the DAPI-DNA complex emits fluorescence. Most of the modern anticancer drugs focus on DNA damage, as it leads to apoptosis and other forms of cell death (Kawanishi and Kiraku 2004; Havelka et al. 2007; Cheung-Ong et al. 2013). This method is mainly used for deducing the DNA damage inside the cells by microscopic visualization. The main principle behind this assay is that the binding of DAPI to the ds-DNA produces a ~20-fold fluorescence enhancement (Barcellona et al. 1990). This is observed due to displacement of water molecules from both DNA as well as DAPI. DAPI also binds to RNA at selective A-U intercalation (Tanious et al. 1992) but the intensity of fluorescence is comparatively low compared to that of DNA. The cells treated with cytotoxic drugs will have nuclear damage (Cheung-Ong et al. 2013), and the fragmented DNA will have clusters of DAPI-DNA complexes that can be distinguished morphologically under a microscopic. Based upon the image, the quantification and a relative dosage-survival analysis can be carried out. As far as the fluorescence characteristic is concerned, the excitation and emission maximum of DAPI (bound to ds-DNA) is 358 and 461 nm, respectively. DAPI is usually excited by a UV lamp, but xenon and mercury-arc lamps can also be used for the excitation.
Stigmasterol alleviates allergic airway inflammation and airway hyperresponsiveness in asthma mice through inhibiting substance-P receptor
Published in Pharmaceutical Biology, 2023
Jimei Zhang, Chonghong Zhang, Li Miao, Zimin Meng, Ning Gu, Guifang Song
According to the design requirements of each group, the cells were fixed with 4% paraformaldehyde for 30 min after different treatments. 0.5% Triton X-100 was used to penetrate cells for 20 min and normal goat serum was used to block non-specific expression at room temperature for 30 min. Diluted primary antibody anti-neurokinin 1 receptor antibody (1:200, bs-0064R, Bioss) was added and incubated overnight at 4 °C. Next day, cells were incubated with secondary antibody Cy3-labelled goat anti-rabbit IgG (H + L) (A0516, 1:200, Beyotime) for 1 h. DAPI was diluted at a ratio of 1:200 and added into cells for 8 min. Finally, an anti-fluorescence quenching agent is used to seal the film to prevent fluorescence quenching. Fluorescence of the substance P antibody was observed under MF52-N fluorescent inverted microscope. Images were taken at the magnification of 200×.
Synthesis characterisation and neuroprotectivity of Silybum marianum extract loaded chitosan nanoparticles
Published in Journal of Microencapsulation, 2023
Hatice Feyzan Ay, Serap Yesilkir-Baydar, Rabia Cakir-Koc
In order to examine the apoptotic effect of H2O2 on cells, DAPI staining method was performed. SH-SY5Y cells that had been exposed to only H2O2 were stained with DAPI. Observed results showed that the number of apoptotic cells was higher for the cells which were exposed to H2O2. Then, as mentioned above, cells that were previously treated with SME were then exposed to H2O2 and incubated for 24 and 48 h respectively. After incubation periods, DAPI staining was performed to both groups. Subsequently, samples were examined under a fluorescent microscope. It was observed that increasing concentrations of SME were able to reduce the number of H2O2-induced apoptotic cells and 20 µg/mL had a number of apoptotic cells significantly similar to control cells (data not shown).
Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies
Published in OncoImmunology, 2022
Ángel Ramírez-Fernández, Óscar Aguilar-Sopeña, Laura Díez-Alonso, Alejandro Segura-Tudela, Carmen Domínguez-Alonso, Pedro Roda-Navarro, Luis Álvarez-Vallina, Belén Blanco
The following mAbs against human proteins were used: PE-conjugated anti-CD2 (clone S5.2), APC-conjugated anti-CD3 (clone UCHT1), and APC-conjugated anti-CD10 (clone HI10a) from BD Biosciences (San Jose, CA, USA) and PC7-conjugated anti-CD19 (clone J4.119) from Beckman Coulter (Marseille Cedex, France). DAPI (SigmaAldrich) was used as a viability marker. Cell surface expression of 19-CAR was analyzed using an APC-conjugated GAM IgG F(ab´)2 (Jackson ImmunoResearch, West Grove, PA, USA). Cell surface-bound 19-BiTE was detected with APC-conjugated anti-His mAb (clone GG11-8F3.5.1, Miltenyi Biotec), and intracellular BiTE was detected with APC-conjugated anti-His mAb after cell fixation and permeabilization with Inside Stain kit (Miltenyi Biotec). Cell acquisition was performed in a BD FACSCanto II flow cytometer using BD FACSDiva software (both from BD Biosciences, San Jose, CA, USA). Analysis was performed using FlowJo V10 software (Tree Star, Ashland, OR, USA).