The cell death extent in spheroids treated with MAL -PTX2S@Pba, BOC -PTX2S@Pba, nPTX2S and Pba for 24 h and exposed to 1 J/cm2 red light was assessed after staining with the LIVE/DEAD® viability/cytotoxicity assay kit (Invitrogen, Milan, Italy). Twenty-four or forty-height hours after irradiation, spheroids were rinsed with PBS and then 150 μL of PBS solution containing 1 μM calcein AM and 4 μM ethidium homodimer-1 (EthD-1) was added to each well for the simultaneous determination of live (calcein positive) and dead (ethidium positive) cells. After 30 min of incubation at 37 °C, spheroids were washed twice with PBS and analyzed with a fluorescence microscope (DMI4000, Leica Microsystems). Acquired images were analyzed by quantifying fluorescence intensity (green and red fluorescence for live and dead cells quantification within the spheroid area, respectively) by using ImageJ software.
Dmi4000
Manufactured by Leica Microsystems
Sourced in Germany
The Leica DMI4000 is an inverted microscope designed for advanced research applications. It features high-quality optics, modular components, and a user-friendly interface. The core function of the DMI4000 is to provide a platform for detailed observation and analysis of samples under high magnification.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Dfc365fx, by Leica Microsystems (2 mentions)
Live dead viability cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Egm 2 medium, by Lonza (1 mentions)
Leica application suite advanced fluorescence las af software, by Leica Microsystems (1 mentions)
Alexa fluor 594 or 488 secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Cell taktm, by BD (1 mentions)
Anti hsf1, by Cell Signaling Technology (1 mentions)
Digital sight ds u1, by Nikon (1 mentions)
Anti e cadherin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Lsrii analyzer, by BD (1 mentions)
Lsk antibodies, by Thermo Fisher Scientific (1 mentions)
15 protocols using dmi4000
1
Assessing Cell Death in Spheroids
2
Evaluating Cell Morphology and PDMS-GL Integrity
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Phase–contrast light microscopy observations were performed in order to evaluate alterations in cell morphology after 48 h of exposure to GL suspensions and to evaluate the integrity of PDMS-GL slices removed from the cell culture medium after the various incubation times. Cells and PDMS-GL slices were visualized with a 10×/0.40 (dry lens) or 5×/0.30 objectives, respectively, using an inverted microscope (DMI4000, Leica Microsystems, Wetzlar, Germany) at room temperature in 1× PBS. The images were captured with a digital camera (DFC365 FX, Leica Microsystems) using LAS-AF 2.0 software (Leica Microsystems).
3
Quantifying EPC Migration Dynamics
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Migration of late EPCs was measured using a modified Boyden chamber assay, as previously described (19 (link)). Briefly, following pretreatment with 20 µmol/l RES for 12 h, EPCs were cultured with TNF-α (10 ng/ml) for another 24 h in a serum-free EGM2 medium. A total of 4×104 EPCs were added in the upper insert of the chamber in an EGM2 medium (Lonza Group, Ltd.,) and also EGM2 medium with fetal bovine serum was added to the bottom chamber. Following incubation for 8 h at 37°C in an incubator with 5% CO2, the membrane in the upper chamber was washed gently with PBS and non-migratory cells were scraped using cotton swabs. Subsequently, EPCs were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with DAPI at room temperature for 15 min, and the number of migratory cells in the bottom chamber in six randomly selected fields/well was calculated under a fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany; DMI 4000; magnification, ×100).
4
Immunofluorescence Staining of FKBP51
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D54 and U251 were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in 1× PBS for 5 min at room temperature. The non-specific block was performed by treating cells with 10% FBS (Biowest), 1% BSA (Biorad), 0.1% in PBS for 30 min at room temperature. Then, the samples were incubated with primary and secondary antibodies. Rabbit polyclonal, FKBP51s and FKBP51 primary antibodies (see Immunoblot and immunoprecipitation section) were used at 1:2000 and 1:50 dilutions, respectively. Alexa Fluor 594 or 488 secondary antibodies were used (1:400; Invitrogen). Nuclei were counterstained with DAPI (Calbiochem-EMD Biosciences, 1:5000). Images were captured with an inverted microscope (DMI4000; Leica Microsystems, Heidelberg, Germany) with Leica Application Suite Advanced Fluorescence (LASAF) software (Leica Microsystems). When required, the brightness, contrast and colour balance of the images were adjusted in Photoshop CS2 (Adobe Systems, San Jose, CA, USA). This adjustment was applied to every pixel in each image.
5
Cytochrome c Translocation Microscopy
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Cytochrome c release from mitochondria to cytoplasm was evaluated using a fluorescence microscope (DMI 4000 B Leica Microsystems, Bannockburn, IL) coupled to the software LAS-AF. The control and 4-NRC-treated cells (1 × 106 cells/mL) were fixed with methanol and then seeded on BD cell-takTM-coated coverslips. Cells were permeabilized and blocked with PBS solution containing BSA (1%, p/v) and Tween 20 (0.1%, v/v) in humidified chamber for 1 h, at room temperature. Then the cells were incubated with cytochrome c antibody for 1 h and nuclei were stained with Hoechst 33258. The cells were then washed three times with PBS and mounted, using an appropriate mounting medium (PBS with 90% glycerol, v/v).
6
Immunofluorescence Staining of Stem Cells
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Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 containing 10% FBS (Invitrogen)/1% BSA in PBS at room temperature for 15 min. Samples were then incubated with primary antibodies overnight at 4 °C. The antibodies used for cell immunofluorescence were against OCT4, NANOG, NESTIN, PAX6, FLAG and H2AK119ub1, respectively. The cells were then washed for four times and 0.1 μg/ml DAPI (Sigma) was included in the final wash to stain nuclei. Images were captured with an inverted microscope (DMI4000, Leica Microsystems).
7
Immunofluorescence Analysis of HSF1 and E-cadherin in Caco-2 Cells
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Caco-2 cells were pretreated with 60 µM ALA for 24 h and exposed to HS for 24 h. Cellular localization of HSF1 and E-cadherin was assessed by an immunofluorescence staining as described previously [16 (link)]. Briefly, after HS exposure, cells were fixed with 10% formalin, washed with PBS, and permeabilized with PBS containing 0.1% Triton-X-100. After blocking in 5% serum, cells were incubated with anti-HSF1 (1:100, Cell Signaling, Danvers, MA, USA) and anti-E-cadherin (1:50, eBioscience) antibodies for 2 h at room temperature followed by incubation with Alexa Fluor conjugated secondary antibodies (Invitrogen). After a nuclear counterstaining with Hoechst 33,342 (1:2000; Invitrogen), the inserts were mounted with FluorSave™ Reagent (Calbiochem) and immune-localization of HSF1 and E-cadherin was determined with a Nikon Eclipse TE2000-U microscope equipped with a Nikon Digital Sight DS-U1 camera using an 40× objective. HSF1-immunostained slides were also examined by a Leica TCS SPE-II confocal laser scanning microscope on a DMI4000 (Leica Microsystems, Wetzlar, Germany); images were acquired with an oilimmersion objective (63×) and assembled using ImageJ 1.47 software.
8
Immunofluorescence Analysis of MUC5AC in NCI-H292 Cells
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Immunofluorescence staining of MUC5AC protein in NCI-H292 cells was carried out according to the previously described method [29 (link)] with some modifications. Briefly, NCI-H292 cells were seeded on an 8-chamber microscope slide and serum-starved for 24 h. After starvation, cells were exposed to A68930 (1 μM), dopamine (1 μM), or CSE (10%) for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed 3 times with PBS. After permeabilization (0.2% Triton X-100 in PBS for 5 min) and blocking (1% bovine serum albumin in 0.1% Triton X-100 in PBS for 15 min), cells were incubated with Alexa Fluor 488-conjugated MUC5AC antibody (sc-21,701 AF488, Santa Cruz Biotechnology) overnight at 4C. After cells were washed twice with PBS, the slide was coverslipped with ProLong gold antifade-reagent with DAPI (Thermo Fisher Scientific), and visualized with an inverted fluorescent microscope (DMI-4000; Leica Microsystems, Wetzlar, Germany). Digitized images were captured with MetaMorph software (Molecular Devices, Sunnyvale, CA). When capturing the images, we kept constant the duration of image capture (300 ms), the image intensity gain, the image enhancement, and the image black level among the samples.
9
Colony-Forming Assay for Hematopoietic Stem Cells
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Cultured HSCs were lifted with TrypLE and resuspended in methylcellulose medium supplemented with murine cytokines (MethoCult GF M3434; Stem Cell Technologies) according to the manufacturer’s protocols. In brief, lifted cells were mixed with methylcellulose medium supplemented with growth factors, dispensed to 35-mm culture dishes using a 3-cm3 syringe, and incubated for 11 to 14 days at 37°C and 5% CO2, at which point the colonies that arose (CFU-GEMM, CFU-GM, CFU-M, CFU-G, CFU/BFU-E, CFU-Mk) were enumerated with an inverted microscope (DMI4000, Leica Microsystems). CFU colonies of more than 30 cells were enumerated. Representative images of the CFU colonies are shown in fig. S9. For the subsequent analysis of colony-forming cells, CFU colonies were harvested in bulk from the methylcellulose medium according to the previously reported protocol (56 ). Harvested colony-forming cells were incubated with CD11b (Mac-1), Gr-1 (Ly-6G/C), or a cocktail of LSK antibodies (eBioscience) and analyzed by flow cytometry (BD LSR II analyzer, BD Biosciences). Flow cytometric analysis of the colony-forming cells indicates that our CFU colony enumeration techniques are consistent with known changes in cell surface antigen expressions (fig. S10).
10
Immunofluorescence Analysis of NANOG
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Cells were washed twice with PBS and then fixed in 4% paraformaldehyde at room temperature for 20 min. After fixation, cells were treated with 0.3% Triton
X-100 in PBS containing 10% goat serum at room temperature for 15 min. Cells were then incubated with primary antibodies at 4°C overnight. The primary antibody used for cell immunofluorescence was anti-NANOG (Novus Biologicals). And then the cells were washed for three times and were incubated with corresponding secondary antibody in a cassette at room temperature for 1 h. Then the cells were washed for three times with PBS and then nuclei were stained with DAPI (Sigma). Images were captured with an inverted microscope (DMI4000, Leica Microsystems).
X-100 in PBS containing 10% goat serum at room temperature for 15 min. Cells were then incubated with primary antibodies at 4°C overnight. The primary antibody used for cell immunofluorescence was anti-NANOG (Novus Biologicals). And then the cells were washed for three times and were incubated with corresponding secondary antibody in a cassette at room temperature for 1 h. Then the cells were washed for three times with PBS and then nuclei were stained with DAPI (Sigma). Images were captured with an inverted microscope (DMI4000, Leica Microsystems).
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