Full‐length wild‐type ECE1c cDNA with in‐frame 5′‐Flag and Myc tags was inserted into the multicloning region of plasmid pLVX‐IRES‐mCherry (Clontech, Mountain View, CA, USA). Site‐directed mutagenesis was performed using the GENEART kit (Invitrogen) according to manufacturer’s instructions. Lenti‐X 293T cells were transfected with 8 μg psPax2 (encoding Gag‐Pol protein), 8 μg pLVX‐IRES‐mCherry (encoding ECE1cWT, ECE1cK6R or empty), and 4 μg pCMV‐VSVg (encoding VSV G‐glycoprotein) and then suspended in 500 μL 0.25 m CaCl2. At 48 h post‐transfection, supernatants containing pseudotyped particles were harvested and passed through a cellulose acetate filter with a pore size of 0.45 μm. Viral particles were purified and concentrated by ultracentrifugation at 150 000 g for 75 min in SureSpin 630 rotor (Thermo Fisher, Vilnius, Lithuania) through a 25% sucrose cushion (TNE‐Sucrose 25%). Finally, cells were cultured at 5 × 104 cells/well in 12‐well plates along with the recombinant lentiviruses at a MOI of 5 under normal growth conditions. Expression of mCherry was examined 72 h post‐transduction under a Nikon Eclipse TS100 Inverted Microscope (Nikon, Tokyo, Japan) equipped with epifluorescence. Cells were expanded for 1 week, and the brightest (mCherry+) cells were sorted on a FACSAria Fusion cell sorter (Becton‐Dickinson, San Jose, CA, USA).
Eclipse ts100 inverted microscope
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Switzerland
The Eclipse TS100 is an inverted microscope designed for routine laboratory use. It features a stable, ergonomic design and provides a magnification range of 40x to 400x. The microscope is equipped with bright-field illumination and a built-in condenser for optimal image quality.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Leukocyte acid phosphatase kit, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Ds fi1 camera, by Nikon (4 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Calcium phosphate coated 48 well plate, by Cosmo Bio (2 mentions)
M csf, by R&D Systems (2 mentions)
Rankl, by R&D Systems (2 mentions)
Cd34 pe, by BioLegend (2 mentions)
Qbsf 60, by Quality Biological (2 mentions)
Cd10 pacific blue, by BioLegend (2 mentions)
Cd19 pe cy5, by BioLegend (2 mentions)
Cd33 apc, by BD (2 mentions)
Methocult h4434 classic, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Plan fluor objective, by Nikon (2 mentions)
Mcwhd3 white light led, by Thorlabs (2 mentions)
Intensilight c hgfi mercury light source, by Nikon (2 mentions)
133 protocols using eclipse ts100 inverted microscope
1
Lentiviral transduction of ECE1c constructs
2
Cell Invasion Assay Using Matrigel
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Cell invasion assay was performed using transparent PET membrane inserts (Corning, NY, USA). In brief, cells in serum free medium were seeded on the upper compartment of the chamber pre-coated with Matrigel (Corning, NY, USA). The lower chamber was filled with medium with 20% FBS as a chemo-attractant. After being incubated for 22 hours, the non-invasive cells on the upper surface were removed by scraping with a cotton swab. Invaded cells on the lower membrane surface were fixed in ethanol (Fisher Scientific, MA, USA) and stained with methylene blue. Cell invasion was quantified by counting invaded cells in each well under Nikon ECLIPSE TS100 inverted microscope (Nikon, Tokyo, Japan) using a 20× objective.
3
Histological Analysis of Testes and Epididymis
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Testes and cauda epididymides were fixed in Bouin’s fixative (41506; Sigma, Waltham, MA, USA) for 24 h. Next, the tissues were dehydrated in graded ethanol and embedded in paraffin. Five-micrometer sections were cut and mounted on glass slides, then stained with hematoxylin and eosin (H&E) or periodic acid schiff (PAS). Finally, images were obtained using a Nikon Eclipse TS100 inverted microscope (Nikon, Tokyo, Japan).
4
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential (ΔΨm) was measured using 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) [35] (link). Following incubation, chondrocytes were loaded with JC-1 (50 µg/ml) in <1, 2, 5, or 21% O2 (37 °C, 40 min) and then washed twice in PBS (preequilibrated at relevant oxygen level) before aliquots of cells were fixed on cover slides (Vector Laboratories, Peterborough, UK). Red and green fluorescence intensity (Ex=490 nm; Em=585 nm for red and Em=514 nm for green) was measured using a Nikon Eclipse TS-100 inverted microscope with Eclipse TS-100/TS-100-F fluorescence attachment (Nikon, Surrey, UK) and intensity ratio calculated using Image J 1.42 software. A reduction in the ratio indicates a mitochondrial membrane depolarization.
5
Senescence-associated β-Galactosidase Assay in Chondrocytes
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Cytochemical staining for senescence associated β-galactosidase activity (SA-β-Gal) was performed for each cell line at three different passages, using the Senescence Cells Histochemical Staining kit (Sigma-Aldrich). After 16 hours of incubation, cells were observed and photographed with a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Europe, Netherlands) coupled to a XM Full HD digital camera (Hangzhou Xiongmai Technologies, China). SA-β-Gal-positive and negative cells were counted on ten random microscope fields, and percentage of senescent cells was calculated. Results were provided as mean percentage of senescent cells and standard error (SE). Primary articular chondrocytes (from donors 4 and 5) were employed as a control and compared with transduced articular chondrocytes.
6
Proliferation and Senescence Evaluation of iMSCs
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Cells were observed with a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Europe B.V., Amsterdam, Netherlands) coupled to a XM Full HD digital camera (Hangzhou Xiongmai Technologies (XM), Hangzhou, China). iMSC proliferation was calculated as cumulative population doublings (PDs) at each passage, following the formula in Equation (1 ), where Nf is the final cell number, Ni is the initial cell number, and log is the natural logarithm [46 (link)]. The number of accumulated generations per days in culture was analysed by regression for each cell line. Generation time was calculated for each cell line at each passage as the number of PDs per day, and generation times of all cell lines were compared.
Histochemical staining for senescence-associated β-galactosidase activity was performed for each cell line after reaching more than 100 PDs at three different passages, using the Senescence Cell Histochemical Staining kit (Sigma-Aldrich Química S.A.). After 16 hours of incubation, β-galactosidase-positive and β-galactosidase-negative cells were counted on ten random microscope fields and percentage of senescent cells was calculated. Results are provided as mean percentage of senescent cells ± standard error. Senescence values of iMSCs and primary MSCs were compared.
Histochemical staining for senescence-associated β-galactosidase activity was performed for each cell line after reaching more than 100 PDs at three different passages, using the Senescence Cell Histochemical Staining kit (Sigma-Aldrich Química S.A.). After 16 hours of incubation, β-galactosidase-positive and β-galactosidase-negative cells were counted on ten random microscope fields and percentage of senescent cells was calculated. Results are provided as mean percentage of senescent cells ± standard error. Senescence values of iMSCs and primary MSCs were compared.
7
Spheroid Invasion Assay with Myogel-Fibrin
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The spheroid invasion assay was done according to Naakka et al. (32 (link)). The UM-HMC-2 cells were seeded at a concentration of 1000 cells per well in 50 µL of the complete medium using a U-shaped ultra-low attachment 96-well plate (Corning, New York, USA) and incubated for four days. Next, the spheroids were embedded in 50 μL Myogel-fibrin gel containing 0.5 mg/mL Myogel, 0.3 U/mL thrombin (Sigma-Aldrich), 33.3 mg/mL aprotinin (Sigma-Aldrich), and 0.5 mg/mL fibrinogen (Merck). After the Myogel-fibrin matrix (30 min) solidification, 100 μL of complete medium was added to the wells.
Images of the spheroids were captured daily using Nikon Eclipse TS100 Inverted Microscope (Nikon, Minato, Tokyo, Japan) at 4x magnification. Analysis of the spheroid invasion area and length of the longest branch was performed using ilastik (freeware) and Fiji ImageJ 1.51 software (33 (link)).
Images of the spheroids were captured daily using Nikon Eclipse TS100 Inverted Microscope (Nikon, Minato, Tokyo, Japan) at 4x magnification. Analysis of the spheroid invasion area and length of the longest branch was performed using ilastik (freeware) and Fiji ImageJ 1.51 software (33 (link)).
8
Isolation and Culture of Primary and Immortalized MEPM Cells
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Primary MEPM cells were isolated from the palatal shelves of wild-type embryos dissected at E13.5 (day of plug considered 0.5 days) in 1x phosphate buffered saline (PBS) and cultured on plastic dishes in medium (Dulbecco’s modified Eagle’s medium (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 50 U/mL penicillin (GIBCO), 50 μg/mL streptomycin (GIBCO) and 2 mM L-glutamine (GIBCO)) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA) as previously described [13 (link)]. Primary MEPM cells were split at a ratio of 1:3 through at most three passages. Immortalized MEPM cells were isolated from Cdkn2a-/- E13.5 embryo palatal shelves as described above and cultured on plastic dishes in medium containing 10% FBS. Immortalized MEPM cells have been split at a ratio of 1:5 through at least 22 passages. Cultured cells were photographed using a Nikon DS-Fi1 color camera (Nikon Instruments Inc., Melville, NY, USA) fitted onto a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Inc.).
9
Cell Invasion Assay with Modifications
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Invasion assays were performed as described in [92 (link)], with a few modifications. A Boyden chamber (CytoSelect 24-well cell invasion assay, Cell Biolabs, San Diego, CA) was used. U251- and U343-transfected cells were harvested using trypsin and counted. Half a million cells were resuspended in serum-free medium. A total of 500 μl of medium containing 10% fetal bovine serum was added to the lower chamber, and 300 μl of resuspended cells was placed in the upper chamber. The assay mixture was incubated at 37 °C and in 5% CO2 atmosphere for 18 h. The plate was removed, and the medium inside the insert was aspirated. The chamber was stained with 1% crystal violet. A microscopic image of the insert was taken with a Nikon Eclipse TS100 inverted microscope equipped with a DS-L2 camera control unit (Nikon Instruments, Inc., Melville, NY) at × 20 magnification. Next, crystal violet was dissolved from stained plates using an SDS solution and optical density was measured with a BioTek Synergy HT microplate reader (BioTek, Winooski, VT) at 570 nm. All experiments were performed with technical triplicates.
10
Cell Microinjection Assay for Caspase-3 Activity
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Cell microinjection (25 (link)) was performed using the Nikon Eclipse TS100 inverted microscope (Nikon Corporation, Tokyo, Japan) equipped with a programmable IM-300 microinjector and an MHW-3 hydraulic micromanipulator (Narishige International, Ltd., London, UK). Microinjection needles were crafted using borosilicate glass capillaries using a P-87 device (Sutter Instrument Co., Novato, CA, USA). Microinjection needles were loaded using Eppendorf microloaders (Eppendorf AG, Hamburg, Germany). Microinjection time and pressure parameters were determined using 0.1% dextran-FITC (3,000 MW) microinjection solution. The optimal injection time was determined as being between 200 and 400 msec, while the optimal microinjection pressure was 6.5 psi. In order to determine the basal activity of C-3, the specific substrate Z-DEVD-R110 was microinjected at a concentration of 1.3 mg/ml into single cells. Images were captured using a DXM-1200C camera (Nikon Corporation).
For each assay, A549 cells were incubated for 3, 6, 12, 18 and 24 h with a 1.85 mM solution of 7-HC in ethanol or 3% ethanol (data for 18 h not shown as the results were not significantly different). Then, culture medium was withdrawn from the Petri dish and the cells were washed twice with 1 ml PBS. Finally, the cells were resuspended in 1 ml PBS and the microinjection of Z-DEVD-R110 into single cells was performed.
For each assay, A549 cells were incubated for 3, 6, 12, 18 and 24 h with a 1.85 mM solution of 7-HC in ethanol or 3% ethanol (data for 18 h not shown as the results were not significantly different). Then, culture medium was withdrawn from the Petri dish and the cells were washed twice with 1 ml PBS. Finally, the cells were resuspended in 1 ml PBS and the microinjection of Z-DEVD-R110 into single cells was performed.
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