To study the effect of statins on preadipocyte proliferation, immortalized murine preadipocytes were plated on 96-well Operetta plates at low density (2.000 cells/cm 2 ) and allowed to attach and recover for 12 hours. Cells were treated for 48h with atorvastatin, cerivastatin, simvastatin, fluvastatin and rosuvastatin (all statins were purchased from Sigma-Aldrich) at two different concentrations (1 and 10mM). Cells were fixed with 4% formaldehyde for 20 min and washed 3 times with PBS. Immediately after washing, cells were stained with Hoechst (Cell Signaling) and Syto60 (Invitrogen), to visualize nuclei and cytosol, respectively. Differentiated adipocytes at day 9 were used for differentiation analysis. Briefly, cells in 96-well optical plate, exposed to statins for the last 48 hours (day 7-9), were fixed with 4% formaldehyde for 20 min and washed 3 times with PBS. Immediately after washing, cells were stained with Bodipy (Invitrogen) for lipid droplets and Hoechst (Cell Signaling) for nuclei. Twenty-five pictures per well were taken with an automated microscope imaging system (Operetta, PerkinElmer). Pictures were analyzed using the Harmony software. In differentiation assay, all cells (Hoechst stained nuclei) surrounded by lipid droplets were considered adipocytes. In proliferation assay, nuclei were counted.
Hoechst
Manufactured by Cell Signaling Technology
Sourced in United States
Hoechst is a fluorescent dye that binds to DNA and is commonly used in cell biology research. It is a cell-permeable dye that emits blue fluorescence when bound to DNA, allowing for the visualization and analysis of cellular nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Bodipy, by Thermo Fisher Scientific (2 mentions)
Operetta, by PerkinElmer (2 mentions)
A1plus confocal microscope, by Nikon (2 mentions)
Rb mcherry, by Abcam (2 mentions)
Goat rb 594, by Thermo Fisher Scientific (2 mentions)
Syto60, by Thermo Fisher Scientific (1 mentions)
Simvastatin, by Merck Group (1 mentions)
Atorvastatin, by Merck Group (1 mentions)
Rosuvastatin, by Merck Group (1 mentions)
Fluvastatin, by Merck Group (1 mentions)
Cerivastatin, by Merck Group (1 mentions)
35 mm glass bottom dishes, by MatTek (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Dfc450 c camera, by Leica (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Cell Signaling Technology (1 mentions)
Transwell insert, by Corning (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Fluoview 10i microscope, by Olympus (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
13 protocols using hoechst
1
Statin Effects on Preadipocyte Proliferation
2
Immunohistochemistry of Adult Ovaries
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For performing immunohistochemistry, 3-day-old female adult ovaries were dissected in phosphate buffer saline (PBS) under a stereoscopic microscope. The ovaries were then fixed in 4% paraformaldehyde (PFA) for 50 min. Later, the samples were washed 3 times with PBST (0.1% Triton X-100 in PBS), each time for 10 min. Blocking was performed in 5% normal goat serum (NGS) for 1 h, and then the samples were incubated with primary antibodies at 4 °C overnight. The following day, PBST was used to wash the samples 3 times and then blocking was again performed in 5% NGS. Later, the secondary antibodies were added and the samples were incubated for 2 h. To stain the DNA, we further used Hoechst (1:5000; Cell Signaling Technology, Danvers, MA, USA). The mounting of ovaries was performed in 90% glycerol. A total of 300 germaria were counted for a single representative experiment of three replicates (100 germaria/replicate).
Among the primary antibodies, we used: mouse anti-α-Spectrin (3A9, AB_528473, 1:100; Developmental Studies Hybridoma Band [DSHB], Iowa City, IA, USA) and rabbit anti-pMad (13820T, 1:400; Cell Signaling Technology), while mouse 488 (1:1000) and rabbit cy3 (1:1000) were used as secondary antibodies. For taking the images, a Nikon A1 plus confocal microscope was used (Nikon, Tokyo, Japan).
Among the primary antibodies, we used: mouse anti-α-Spectrin (3A9, AB_528473, 1:100; Developmental Studies Hybridoma Band [DSHB], Iowa City, IA, USA) and rabbit anti-pMad (13820T, 1:400; Cell Signaling Technology), while mouse 488 (1:1000) and rabbit cy3 (1:1000) were used as secondary antibodies. For taking the images, a Nikon A1 plus confocal microscope was used (Nikon, Tokyo, Japan).
3
Live-cell Imaging of Peptide Cross-presentation
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For live cell imaging of peptide cross-presentation, NBS fibroblasts were grown on 35 mm glass bottom dishes (MatTek Life Sciences, MA, USA) coated with poly-D-lysine. Hoechst (Cell signaling, Leiden, The Netherlands; 0.5 µg/mL) and Lysotracker deep red (Life Technologies; 0.1 µM added 15–30 min before imaging) were used to stain the nucleus and the lysosomes for detection by confocal microscopy. After addition of internally quenched peptide (20 µg/mL), time-lapses were collected on a Dragonfly spinning disk microscope (Oxford instruments, Abingdon, UK) adapted with a climate control chamber. Images were acquired using Hcx PL 63×1.32 oil objectives (Leica Camera, Solms, Germany), and data were analyzed using Fiji software.
4
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis was performed in a 5 laser LSR Fortessa (BD Bioscience) flow cytometer. DNA was stained with HOECHST (Cell Signalling) and viability assessment was performed with propidium iodide staining. After harvesting cells as described above for the modulation of cisplatin effects in NTera2 cells section, cells were resuspended in a 15 μg/μl HOECHST (diluted in 2%FCS DPBS) solution and incubated in the dark for 30 min at 37 °C prior to cell cycle analysis.
5
Fluorescent Imaging of SaOs2 Cell Actin
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Cells grown on all samples were examined by means of fluorescence imaging. After 24 h and 72 h of culture, SaOs2 cells were fixed with 4% paraformaldehyde at room temperature and kept in phosphate-buffered saline (PBS) at 4 °C before labeling. The fixed cells were then permeabilized with 0.2% TritonX-100 and blocked in 0.5% bovine serum albumin. In order to visualize the actin filaments, the cells were stained with Alexa Fluor 488-conjugated Phalloidin (Cell Signaling, Technology, Danvers, MA, USA). The cells were then treated with 1 μg/mL Hoechst (Cell Signaling, Technology, Danvers, MA, USA) in order to label the nuclei. After each incubation, the samples were washed three times with PBS. In the end, the specimens were analyzed on glass slides using a DM 4000 B LED fluorescence microscope equipped with a DFC 450 C camera (Leica Microsystems, Wetzlar, Germany) with appropriate filters.
6
Mature Adipocyte Differentiation Analysis
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Mature beige adipocytes at day 18 after GPR180 silencing were used for differentiation analysis. Briefly, cells in 96-well optical plate were fixed with 4% formaldehyde for 20 min and washed 3 times with PBS. Immediately after washing, cells were stained with Bodipy (Invitrogen) for lipid droplets and Hoechst (Cell Signaling) for nuclei. Twenty-five pictures per well were taken with an automated microscope imaging system (Operetta, PerkinElmer). Pictures were analyzed using the Harmony software v3.5. In differentiation assay, all cells (Hoechst stained nuclei) surrounded by lipid droplets were considered adipocytes.
7
Uptake Kinetics of RJ Extracellular Vesicles
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Release patterns found with NTA analyses were verified using an EV uptake assay. Cells
were analyzed on days 1, 3, and 7. For each analysis, 3T3-L1 cells were seeded at a
concentration of 1.25 × 104 cells/cm2 and left to adhere overnight
to ensure equal confluence. Microvesicles were stained with CFSE membrane dye
(Carboxyfluorescein succinimidyl ester; Thermo Fisher, US) as described previously (Schuh
et al., 2019 (link)), and incorporated into 2 mg/ml
collagen gels at a concentration of 2.5 × 109/ml. Gels containing CFSE-stained
RJ EVs were co-localized with the cells using a Transwell insert (6.5 mm diameter, 8 µm
polycarbonate membrane; Corning, US). CFSE-stained RJ EVs added directly into the media
served as positive control. After overnight incubation, cells were washed 3× with pf-PBS
to remove residual RJ EVs and fixed with 4% formaldehyde for 30 min. Nuclei were stained
with Hoechst (Cell Signaling, US) and samples were washed 3× with PBS prior to mounting.
Images were taken on an Olympus Fluoview 10I microscope.
were analyzed on days 1, 3, and 7. For each analysis, 3T3-L1 cells were seeded at a
concentration of 1.25 × 104 cells/cm2 and left to adhere overnight
to ensure equal confluence. Microvesicles were stained with CFSE membrane dye
(Carboxyfluorescein succinimidyl ester; Thermo Fisher, US) as described previously (Schuh
et al., 2019 (link)), and incorporated into 2 mg/ml
collagen gels at a concentration of 2.5 × 109/ml. Gels containing CFSE-stained
RJ EVs were co-localized with the cells using a Transwell insert (6.5 mm diameter, 8 µm
polycarbonate membrane; Corning, US). CFSE-stained RJ EVs added directly into the media
served as positive control. After overnight incubation, cells were washed 3× with pf-PBS
to remove residual RJ EVs and fixed with 4% formaldehyde for 30 min. Nuclei were stained
with Hoechst (Cell Signaling, US) and samples were washed 3× with PBS prior to mounting.
Images were taken on an Olympus Fluoview 10I microscope.
8
Immunofluorescence Staining of MSK1 and NeuN
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Sections were washed with PBS and then blocked (2 h room temperature) with 10% normal goat serum in PBST. Next, sections were incubated overnight (at 4℃) with a rabbit polyclonal total MSK1 antibody (1:500 dilution, Cell Signaling, catalog number: 3489) and with a mouse monoclonal anti-NeuN antibody (1:1,000 dilution, Millipore, Billerica, MA; catalog code: MAB377). Tissue was then washed 5× in PBST and incubated for 2 h (at 22℃) with goat polyclonal Alexa 488- and donkey polyclonal Alexa 594- (1:1,000 dilution, Invitrogen, Carlsbad, CA) conjugated secondary antibodies. Next, sections were washed, and DNA was labeled with Hoechst (1 µg/ml: Cell Signaling). Finally, tissue was mounted with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI), and images were acquired with a Leica SP8 confocal microscope.
9
Immunocytochemical Analysis of Transduced MSCs
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Transduced MSCs were washed with PBS and fixed using 10% formalin fixation for 10 min on ice. MSCs were blocked in 10% SEABLOCK (Thermo Fisher Scientific, Catalog #37527) + PBS + 0.1% Triton X100 (VWR, Catalog #0694-1L) for 1 h prior to incubation with primary antibody 1:500 rb-mCherry (abcam, Boston, MA, United States, Catalog #ab167453) and 1:1,000 ms-Nestin (abcam, Catalog# 22035) for 1.5 h. MSCs were rinsed 3× with ice cold PBST and incubated with AlexaFluor secondary antibody 1:1,000 goat rb-594 (Thermo Fisher Scientific, Catalog #A32740) and 1:1,000 goat ms-488 (Thermo Fisher Scientific, Catalog #A28175) for 1.5 h and then with 1:1,000 Hoechst (Cell Signaling, Catalog #4082) for 10 min. MSCs were then rinsed 2× with PBST before storing in PBS. Cells were imaged on a Nikon Eclipse Ti-U inverted research microscope (Nikon, Melville, NY, United States).
10
Immunofluorescent Staining of Neural Tissues
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Sagittal sections were incubated in a 10% SEABLOCK + 0.1% TBST blocking solution for 1 h prior to incubation with 1:500 rb-mCherry (abcam, Boston, MA, United States, Catalog #Ab167543) and 1:1,000 gp-NeuN (Sigma-Aldrich, St. Louis, MO, United States, Catalog #AB90) at 4°C overnight on gentle agitation. Tissue was then washed with TBST three times for 5 min before incubation with 1:1,000 goat rb-594 and 1:1,000 goat gp-647 AlexaFluor secondary antibodies (Thermo Fisher Scientific, Waltham, MA, United States, Catalog #A111012 and A21450), and then labeled with 1:1,000 Hoechst (Cell Signal Technology, Beverly, MA, United States, Catalog #4082) for nuclear visualization for 5 min. Tissue was then washed with TBST 2× prior to a final TBS 1× wash before mounting and coverslipped with Fluoromount (Sigma-Aldrich, St. Louis, MO, United States, Catalog #F4680-25ML).
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