Nanomechanical Analysis of Cells from Cancer Patients
Lajos P. Balogh in Nano-Enabled Medical Applications, 2020
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.
Structure-Function Elucidation of Flavonoids by Modern Technologies
Dilip Ghosh, Pulok K. Mukherjee in Natural Medicines, 2019
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.
Sperm chromatin assessment
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
Aliquots of sperm at a concentration of 5-10 million/mL are prepared by diluting in PBS. The samples are mixed with 1% low-melting point aqueous agarose (to obtain a 0.7% final agarose concentration) at 37°C. Aliquots of 50 μL of the mixture are pipetted onto a glass slide precoated with 0.65% standard agarose dried at 80°C, covered with a coverslip, and left to solidify at 4°C for four minutes. The coverslips are then carefully removed, and the slides are immediately immersed horizontally in a tray of freshly prepared acid denaturation solution (0.08 N HCl) for seven minutes at 22°C in the dark, which generates restricted single-stranded DNA motifs from DNA breaks. Denaturation is then stopped, and the proteins are removed by transferring the slides to a tray with neutralizing and lysing solution 1 (0.4 mol/L Tris, 0.8 mol/L DTT, 1% SDS, and 50 mmol/L EDTA, pH 7.5) for 10 minutes at room temperature. The slides are then incubated in neutralizing and lysing solution 2 (0.4 mol/L Tris, 2 mol/L NaCl, and 1% SDS, pH 7.5) for five minutes at room temperature. The slides are thoroughly washed in Tris-borate EDTA buffer (0.09 mol/L Tris-borate and 0.002 mol/L EDTA, pH 7.5) for two minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (two minutes each), and air-dried. Cells are stained with DAPI (2 μg/mL) for fluorescence microscopy (Figure 6.2a) (149).
Anti-oxidative effect of the tyrosine kinase inhibitor nintedanib: a potential therapy for chronic lung allograft dysfunction?
Published in Experimental Lung Research, 2020
Elke Boxhammer, Karla Lehle, Christof Schmid, Marietta von Suesskind-Schwendi
Tissue sections were fixed, blocked, and heated as mentioned above. Afterwards sections were incubated with tris-buffered saline (TBS) buffer containing 2% normal donkey serum (NDS) (60 min, RT). After a rinsing process with TBS, primary antibodies were incubated for 16 hours at 4 °C. Therefore, Vimentin- (Santa Cruz sc6260, Dallas, US; diluted 1:400) and ED 2/CD 163-antibody (BioRad MCA342GA, Hercules, US; diluted 1:100) were combined with TGM-2-antibody (Novus Biologicals NBP2-44595, Littleton, US; diluted 1:50), whereas CD 31-antibody (Novus Biologicals NB100-2284; diluted 1:100) was merged with TGM-2-antibody (Invitrogen PA5-95256, Carlsbad, US; diluted 1:200). Tissue sections again were washed a few times with TBS and subsequently treated with fluorescent secondary antibodies (Dianova Alexa-Fluor 594 711-585-152, Alexa-Fluor 488 711-545-152, Alexa-Fluor 488 715-585-150, Alexa Fluor 594 715-545-15-1, Hamburg, Germany; diluted 1:300). Staining with DAPI in a dilution of 1:200 was done. Finally, the slides were covered in fluoromount and examined under fluorescent light.
Non-invasive targeted iontophoretic delivery of cetuximab to skin
Published in Expert Opinion on Drug Delivery, 2020
Maria Lapteva, Marwa A. Sallam, Alexandre Goyon, Davy Guillarme, Jean-Luc Veuthey, Yogeshvar N. Kalia
Image acquisition was performed in the Bioimaging Core Facility, Faculty of Medicine, University of Geneva. Skin sections were examined with an Axio Imager.Z1 Microscope (Carl Zeiss; Jena, Germany) equipped with an HXP-120 xenon lamp and TL Halogen Lamp. Excitation and emission wavelengths for (i) DAPI were 353 and 465 nm and (ii) Alexa488 were 493 and 517 nm, respectively. Images were acquired using an Axiocam 506 camera. Typical exposure times were 200 ms for DAPI and 2 s for Alexa488 when 10x objective was used (EC Plan-Neofluar 10x/0.30 M27). Lower exposure times were used for objectives with higher magnification (EC Plan-Neofluar 20x/0.5 and Plan Apochromat 63x/1.4 oil DIC). All images acquired with the same objective used the same exposure time, lamp intensity and gray pixel setting in order to allow accurate comparison of signals. Images were acquired using Zen 2.3 Pro software and processed using Image J 1.52 n software.
Dual drug-loaded cubic liquid crystal gels for transdermal delivery: inner structure and percutaneous mechanism evaluations
Published in Drug Development and Industrial Pharmacy, 2019
Xiaoqin Chu, Xingqi Wang, Chunling Tian, Liu Liu, Mengqiu Xia, Jianqin Jiang, Shuangying Gui
SH (purity > 98.0%) was obtained from Chengdu Mansite Biotechnology Co., Ltd. (Chengdu, China). CA (purity > 98.0%) was brought from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). PT (purity > 95.0%) was acquired from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). Fluorescein sodium was purchased from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). 40,6-diamido-2-phenylindole (DAPI) (purity > 98.0%) was obtained from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). 4% paraformaldehyde was provided by Hefei Bayer Chemical Technology Co., Ltd. (Heifei, China). Dialysis bag (molecular weight cutoff: 1000 Da) was purchased from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). Urethane (purity > 99.6%) was purchased from Shanghai TargetMol Biotechnology Co., Ltd. (Boston, USA). Purified water was kindly prepared by Xinyi Jinzhu Chemical Industry Co., Ltd. (Shanghai, China). All other chemicals and reagents were of analytical purity or higher except that methanol were of HPLC grade in the study.
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