Fibroblasts were plated on a 24 well plate, seeded at 10^5 cells per ml (Thermo Scientific) the day before the experiment and allowed to reach full confluence. A scratch wound was generated with a yellow pipette tip and cells were washed once with fresh complete media containing 1 μg/ml Hoechst before setting up points for imaging. Prior to imaging, the media containing Hoechst (Sigma) was replaced with complete media or media with different dilutions of BCM containing 1 μg/ml of propidium iodide (PI) and 1 μg/ml Hoechst. Time-lapse imaging was performed in an incubation chamber at 37°C in presence of 5% CO2 with a Leica DMI 6000. The distances migrated were analyzed with Image J software (NIH) plugin by manually drawing the leading edge of each time point. Conversion of migration distance from pixels to μm was performed. Image J software (NIH) plugin was also used to determine the total number of cells (Hoescht positive) and total number of dead cells (PI positive) in each image. At least two areas each (wound and non-wound) were taken for each well and three technical replicates for each experiment was performed.
Hoechst
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Hoechst is a line of laboratory equipment manufactured by Merck Group. It is designed to provide reliable and consistent performance for various scientific applications.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (29 mentions)
Triton x 100, by Merck Group (28 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (27 mentions)
Lsm 710, by Zeiss (21 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Propidium iodide, by Merck Group (16 mentions)
Paraformaldehyde (pfa), by Merck Group (15 mentions)
Propidium iodide, by Thermo Fisher Scientific (14 mentions)
Lsm 710 confocal microscope, by Zeiss (11 mentions)
Mowiol, by Merck Group (10 mentions)
Lsm 880, by Zeiss (10 mentions)
Lsm 700, by Zeiss (10 mentions)
Axio observer z1, by Zeiss (10 mentions)
Lsm 880 confocal microscope, by Zeiss (10 mentions)
Vectashield, by Vector Laboratories (9 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (9 mentions)
Fluoromount g, by Southern Biotech (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Sp2 confocal microscope, by Leica (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
711 protocols using hoechst
1
Scratch Wound Healing Assay with Fibroblasts
2
Assessing Neuronal Viability and Outgrowth
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The Hoechst/PI assay and total neurite outgrowth assessment were conducted according to a previous study (14 (link)). For the Hoechst/PI assay, Hoechst (Sigma-Aldrich; Merck KGaA) and PI (Sigma-Aldrich; Merck KGaA) were added to the medium at 37°C followed by incubation for 30 min. Images were acquired using an inverted fluorescence microscope (Leica Microsystems GmbH), and the cell apoptotic rate was calculated by counting the total cells and damaged cells. For the total neurite outgrowth assessment, the cellular morphology was observed on a fluorescence microscope (Leica Microsystems GmbH; magnification, ×200) in 5 randomly selected fields of view. The neurite outgrowth of each cell was measured by imaging software Image Pro Plus (v6.0; Media Cybernetics, Inc.), and the total neurite outgrowth per cell was calculated as follows: total length of neurite outgrowth / number of included cells.
3
Zebrafish Embryo Labeling Protocol
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Wild-type (wt) adult zebrafish (Danio rerio) were purchased from a commercial source and were kept at 28 °C at 14 hr of light and 10 hr of dark per day. Fish were fed three times daily and were crossed as shown in the “Zebrafish Book”36 . Fertilized eggs were chosen under a stereomicroscope (Stereo Discovery.V8, Zeiss Microscopy) and collected at 4 hours post fertilization (hpf). Healthy embryos were maintained in zebrafish embryo medium (i.e. NaCl, KCl, CaCl2.2H2O and MgCl2.6H2O; pH 7.3) and housed in an incubator at 28 °C.
Staining, Hoechst or DiAsp: For live staining, dechorionated zebrafish embryos at 18–22 hpf were incubated with 5 μg/ml of Hoechst (33342, Sigma) or 2 mg/ml of 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (DiAsp, Sigma) in embryo medium for 15 min in the dark. Labeled embryos were rinsed three times with fresh embryo medium, immobilized on 0.5% agarose gel, and placed on a petri dish. The petri dish was filled with fresh embryo medium at 28 °C for imaging. All animal experiments were performed in full compliance with the revised directive 2010/63/EU, Italian Legislation (art. 31 D.lgs.26/2014) and the methods were carried out in “accordance” with the approved guidelines.
Staining, Hoechst or DiAsp: For live staining, dechorionated zebrafish embryos at 18–22 hpf were incubated with 5 μg/ml of Hoechst (33342, Sigma) or 2 mg/ml of 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (DiAsp, Sigma) in embryo medium for 15 min in the dark. Labeled embryos were rinsed three times with fresh embryo medium, immobilized on 0.5% agarose gel, and placed on a petri dish. The petri dish was filled with fresh embryo medium at 28 °C for imaging. All animal experiments were performed in full compliance with the revised directive 2010/63/EU, Italian Legislation (art. 31 D.lgs.26/2014) and the methods were carried out in “accordance” with the approved guidelines.
4
Isolation and analysis of fetal liver cells
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E11.5–13.5 embryos were harvested and dissected to collect the FL. Hundred microlitre of PBS containing 1 million single cells obtained from whole E13.5 FLs were stained with 5 ng μl−1 CD71-FITC (553266) and Ter119-PE (553673) antibodies (BD Pharmingen). Hoechst was used as a viability dye (Sigma), and cells positive for Hoechst staining were excluded from further analysis (>70% of the total cell population consisted of viable cells). Flowcytometric analysis was performed using a BD LSRFortessa flowcytometer (BD Biosciences), collecting a minimum of 10,000 events per sample. FACS sorting was performed with a BD FACSAria III (BD Biosciences) using the above described staining protocol. At least 1 million cells were sorted for RNA extraction.
5
TUNEL Assay for Apoptosis Detection
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To localize cells undergoing nuclear DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using in situ apoptosis detection kit (Roche, Branchburg, NJ, USA). Cells were fixed with para-formaldehyde. After the cells had been washed with phosphate-buffered saline (PBS), the TUNEL assay was performed according to the manufacturer’s protocol with the fluorescein conjugated probe. The cells were counter stained with Hoechst to assess their nuclear morphology. The Hoechst staining was performed by incubatingthe slides for 5 min at room temperature with 1 mg/mL Hoechst (Sigma) diluted in PBS. The slides were washed and mounted for analysis under a fluorescent microscope (ZEISS, Jean, Germany).
6
Immunofluorescence Staining of Keratinocytes
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Immunofluorescence (IF) staining was used to detect keratinocyte proliferation and differentiation. For IF staining of primary keratinocytes, cells were briefly fixed with 4% paraformaldehyde (4% PFA) for 15 min, rinsed with phosphate-buffered solution (1×) (PBS, 1st Base, Singapore), blocked with 10% donkey serum (Sigma, USA) and incubated at 4°C overnight with primary antibodies. Slides were then rinsed and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
For IF staining of paraffin sections, standard dewaxing and rehydration steps were performed. Antigen retrieval was performed using citrate buffer pH6 (Novus Biologicals, USA) and 2100 Antigen Retriever (ProteoGenix, France), followed by Proteinase K (Sigma, USA) digestion for 15 min at room temperature. Sections were rinsed with PBS and blocked with donkey serum (Sigma, USA), and then incubated with primary antibodies overnight at 4°C. Next, the slides were rinsed in PBS-T (0.05% Tween-20, BioBasic, Singapore) and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, and then rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
For IF staining of paraffin sections, standard dewaxing and rehydration steps were performed. Antigen retrieval was performed using citrate buffer pH6 (Novus Biologicals, USA) and 2100 Antigen Retriever (ProteoGenix, France), followed by Proteinase K (Sigma, USA) digestion for 15 min at room temperature. Sections were rinsed with PBS and blocked with donkey serum (Sigma, USA), and then incubated with primary antibodies overnight at 4°C. Next, the slides were rinsed in PBS-T (0.05% Tween-20, BioBasic, Singapore) and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, and then rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
7
Determining Cell Viability with EdU Assay
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EdU assay was adopted to determine cell viability. Briefly, following treatment with 200 μl of 5-ethynyl-20-deoxyuridine at room temperature for 2 h, the cells were fixed in 4% triformol for 30 min and were treated with 0.4% Triton X-100 for 10 min and 100 μl of Apollo reagent for 40 min. After staining by Hoechst (Sigma Aldrich, USA), the cell viability was determined according to the ratio between Hoechst-positive cells (blue) to EdU-positive cells (red).
8
Visualizing PAK4-Mediated Cell Adhesion
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MCF7 cell line stably overexpressing FLAG-PAK4 were re-plated for 30 min onto 20 μg/ml collagen type I coated dishes in adhesion buffer (RPMI 1640, 2 mM CaCl2, 1 mM MgCl2, 0.2 mM MnCl2 and 0.5% BSA). Cells were fixed with 3 % paraformaldehyde for 10 min, permeabilized with 0.1% Triton-X for 5 min, blocked with 2 % BSA for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Anti-FLAG mab (F3165) were obtained from Sigma. The anti-N-WASP (HPA005750) antibody was obtained from Atlas Antibodies. F-actin was labeled with siR-actin at 1 μM for 1 h (SC001, Cytoskeleton, Inc) and nuclei were stained with 8 μM Hoechst (14533, Sigma). For cell morphology, H1299 cells were attached overnight on a vitronectin (10 μg/ml) coated dish 3 d after siRNA transfection. Cells were fixed, permeabilized and blocked at room temperature. The nuclei were stained with 8 μM Hoechst (14533, Sigma) and F-actin with 0.2 μM Alexa Fluor 568 conjugated phalloidin for 30 min (A12380, Life Technologies). Images were acquired with a Nikon A1 confocal microscope using an oil immersion objective (60X/1.4 NA) and the NIS software.
9
Histological Analysis of Neural Connections
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Deeply anesthetized animals were transcardially perfused with 0.01 M PBS (pH 7.4) followed by 4% paraformaldehyde. Brains were post-fixed for 6 hr, cryoprotected with 30% sucrose solution in 0.1 M PBS, and serially cut in coronal sections (thickness: 40–50 µm) using a HM 450 Sliding Microtome (Thermo Fisher). Sections were counterstained with Hoechst (1:300, Sigma–Aldrich, Milan, IT), mounted, and coverslipped with a DABCO [1,4-diazobicyclo-(2,2,2)octane]-based Antifade Mounting Medium. Fluorescence images were acquired with a Leica SP5 inverted confocal microscope (Leica Microsystems, Milan, IT).
For the evaluation of the anatomical connections between VPM and S1bf, mice were perfused after 10 days from the injection and GRIN lens implantation. 50 µm thick coronal brain slices were cut, counterstained with Hoechst (1:300, Sigma–Aldrich, Milan, IT), and mounted with an Antifade Mounting Medium (Vectashield, Burlingame, CA). Confocal images were acquired with a Nikon Eclipse scope (Nikon, Milan, IT).
For the evaluation of the anatomical connections between VPM and S1bf, mice were perfused after 10 days from the injection and GRIN lens implantation. 50 µm thick coronal brain slices were cut, counterstained with Hoechst (1:300, Sigma–Aldrich, Milan, IT), and mounted with an Antifade Mounting Medium (Vectashield, Burlingame, CA). Confocal images were acquired with a Nikon Eclipse scope (Nikon, Milan, IT).
10
Evaluating COV434 Cell Viability
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COV434 cells, divided into Control, LPS (1μg/mL, 5μg/mL, 10μg/mL,Sigma, USA), Bay 11-7082 (5μM, 10μM,calbiochem, Germany), were seeded into 96-well plates. These cells (1 × 10 5 cells/mL) were maintained in RPMI Medium 1640 basic (1×) + 10% fetal bovine serum at 37 °C and 5% CO2. The cell viability was assessed using CCK8 assay (cholecystokinin-8). Briefly, 10 μl of CCK8 reagent (Dojindo, Kumamoto, Japan) was added to the 96-well plates and incubated for 12h, 24h and 48h at 37 °C. The absorbance values were measured at 450 nm using a Bio-Rad model 450 microplate reader (Bio-Rad, USA). The cell viability was indirectly determined by examining the ratio of the absorbance value of LPS-treated cells, and Bay 11-7082-treated cells relative to the control cells.For Hoechst (1:1000, Sigma, USA) / Propidium Iodide (PI, 1:1000, Sigma, USA) staining, the cells were cultured and washed twice with cold PBS, and then incubated with Hoechst/PI for 45 min at 37°C in the dark.
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