Male mouse embryonal (E7) carcinoma P19 cells60 (link) (ATCC, CRL-1825) were maintained in Dulbecco’s modified Eagle medium with 4,500 mg/L of glucose supplemented with 100 units/mL of penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (FBS, Gemini Bio-Products). For culture, cells were incubated at 37°C and 5% CO2 atmosphere. All experiments have been conducted with original ATCC cells not exceeding eight passages. To induce neuronal differentiation, 3 x 105 cells were seeded in poly-L-lysine-coated 6-well plates in TaKaRa NDiff® 227 Medium (Takara) with retinoic acid (500 nM; Sigma) as previously described.61 (link) Media was changed daily up to 6 days after induction. Morphological changes were visualized using the brightfield channel of the Nikon Eclipse TS2-fl microscope (Nikon Instruments Inc.).
Eclipse ts2 fl microscope
Manufactured by Nikon
Sourced in United States, Germany
The Eclipse Ts2-FL microscope is a high-quality research-grade microscope designed for fluorescence imaging applications. It features a stable and ergonomic design, providing reliable performance for a wide range of microscopy techniques.
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Lab products found in correlation
Ds fi3 camera, by Nikon (4 mentions)
Ndiff 227 medium, by Takara Bio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Stempro differentiation kit, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Emetine, by Merck Group (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Eclipse ts2 inverted microscope, by Nikon (1 mentions)
Ds qi2, by Nikon (1 mentions)
Eclipse ti2 e, by Nikon (1 mentions)
Celltiter 96 non radioactive cell proliferation assay mtt, by Promega (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
12 protocols using eclipse ts2 fl microscope
1
Neuronal Differentiation of Mouse P19 Cells
2
Trilineage Differentiation Potential of MSCs
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A trilineage differentiation assay was used to evaluate the trilineage differentiation potential of MSCs to give rise to chondrocytes, osteoblasts and adipocytes using a commercially available differentiation media (StemPro™ Differentiation Kits, Thermo Fisher Scientific). For chondrogenic differential assay, cells were plated at a density of 106 cells/well in ultra-low attachment 96-wellplate and then induced using the chondrogenic differentiation media after 24 h. The differentiation media were replaced twice weekly. After 23 days, differentiation was assessed by Alcian Blue solution staining of sulfated proteoglycans. For osteogenic and adipogenic differential assays, cells were plated at a density of 100,000 cells/well in 24well-plates and replaced with corresponding differentiation media the next day. After 16 days, adipogenic differentiation was assessed by Oil Red O solution staining of oil droplets. For osteogenic differentiation, differentiation was assessed by Alizarin red S solution staining of calcium deposits at day 23 post induction. Images were taken at 4 × objective for chondrogenesis and 20 × objective for both osteogenesis and adipogenesis using a Nikon Eclipse Ts2-FL microscope (Nikon Instruments Inc., NY, USA).
3
Measuring MSC Proliferation and Metabolic Activity
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For estimation of the generation time, population doublings in passages 2 and 3 were evaluated. MSC were seeded at a density of 3,000 cells/cm2 and incubated for 5 days, with a medium change after 3 days. Phase-contrast photomicrographs were obtained at standardized settings using a Nikon Eclipse Ts2-FL microscope with a DS-Fi3 camera (Nikon GmbH, Duesseldorf, Germany) at day 5 before passaging. Cells were then trypsinized and counted using a hemocytometer and trypan blue for exclusion of dead cells. The generation times were calculated using the following formula:
Using Fiji ImageJ software, the images obtained before passaging were uniformly enhanced in contrast, a background subtraction was done and images were binarized, with adapted thresholds for each image, and the confluent area within each image was measured. In addition, MSC metabolic activity was measured at day 1 and 5 using a tetrazolium compound (MTS) assay according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Mannheim, Germany). The mean absorbance at day 5 was divided by the mean absorbance at day 1 as an indicator for metabolic activity.
Using Fiji ImageJ software, the images obtained before passaging were uniformly enhanced in contrast, a background subtraction was done and images were binarized, with adapted thresholds for each image, and the confluent area within each image was measured. In addition, MSC metabolic activity was measured at day 1 and 5 using a tetrazolium compound (MTS) assay according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Mannheim, Germany). The mean absorbance at day 5 was divided by the mean absorbance at day 1 as an indicator for metabolic activity.
4
Diverse Cellular Stress Responses
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1 × 106 cells in DMEM Full Media were seeded in six-well plate format. Forty-eight hours later, media was replaced by 2 ml DMEM Full Media containing a either 700 μg/ml hygromycin B (HygroB) (10687010, Thermo Fisher), 700 nM emetine (E2375, Merck), 100 μg/ml blasticidin S (BlaS) (R21001, Thermo Fisher), 100 μg/ml cycloheximide (CHX), 50 mM CaCl2 (1.02391, Merck), 0.45 M Sucrose (84097, Merck), or DMEM only (no FBS; starvation). Cells were visualised in an Eclipse Ts2-FL microscope (Nikon). Results depicted on Figure 5A were obtained at 2 h (Sucrose), 18 h (BlaS and CHX), 20 h (emetine and HygroB) and 24 h (CaCl2 and starvation) of treatment. Every condition had its own ‘untreated control’ per cell line; Figure 5A shows a representative untreated control. Activation of the UPR (Figure 3D ) was performed by treating cells with 2 μM thapsigargin (T9033, Merck) for 3 h. Proteins were then extracted with RIPA buffer containing 1 mM Na3VO4, 5 mM Na4P2O7 and 50 mM NaF to retain their phosphorylation status.
5
Quantifying Cell Adhesion in HeLa Cells
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HeLa cells at a density
of 25 × 103 cells/well were pretreated with an inhibitor
for 15 min before being plated onto a gelatin-coated 24-well plate
and were incubated for 30 min at 37 °C. Further, 4% paraformaldehyde
was used to fix the attached cells to the surface.63 (link) A Nikon ECLIPSE
TS2-Fl microscope was used to capture the microphotographs,
and the number of attached cells was quantified using NIH Image J
software. Based on these images, differential analysis was performed
for treated and untreated conditions.63 (link)
of 25 × 103 cells/well were pretreated with an inhibitor
for 15 min before being plated onto a gelatin-coated 24-well plate
and were incubated for 30 min at 37 °C. Further, 4% paraformaldehyde
was used to fix the attached cells to the surface.63 (link) A Nikon ECLIPSE
TS2-Fl microscope was used to capture the microphotographs,
and the number of attached cells was quantified using NIH Image J
software. Based on these images, differential analysis was performed
for treated and untreated conditions.63 (link)
6
Microfluidic Emulsion Imaging Protocol
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After amplification, the samples were retrieved by a 1 ml plastic syringe connected with tubing (PE60, Scientific Commodities Inc.). The samples were then manually infused into a microfluidic channel where a stream of spacing oil was maintained by infusing oil at 300 µl h−1 using a syringe pump (LSP01‐2A, Longer Precision Pump Co.) to ensure reasonable spacing between emulsions. The microfluidic channel had a width of 800 µm and a height of 46.54 ± 1.31 µm, as measured by cutting through a PDMS slab and imaging under a microscope. The flow of emulsions was imaged in brightfield mainly using an inverted microscope (Eclipse Ti2‐E, Nikon) coupled with a camera (DS‐Qi2, Nikon). Videos were captured at a frame rate of 20 frames per second and lasted for 3–5 min, resulting in about 5400–9000 frames for each video. When studying the dependence of the algorithm on image quality, another two imaging systems were used, including an Eclipse Ts2 inverted microscope (Nikon) coupled with a Phantom VEO‐E310L camera (Vision Research) and an Eclipse Ts2‐FL microscope (Nikon) coupled with a DS‐Fi3 camera (Nikon). Fluorescence of DNA‐intercalating reagents was imaged using the Eclipse Ti2‐E microscope.
7
Microscopic Examination of Cell Morphology
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Cells were examined for morphology using a Nikon Eclipse Ts2-FL microscope equipped with a Nikon DS-Fi3 camera. Images and scaling were captured using the NIS-Elements imaging software version 5.30.05.
8
Wound Healing Assay for Cell Migration
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Wound-healing assay was applied to assess cell migration. siRNA-transfected cells were seeded on a 6-well plate. After 24 h, cells were treated with DMSO (control) or rapamycin without FBS. Once cells are confluent, a clear wound line was created using a sterile 200 μl pipette tip. Cell images containing the wound area were taken at 0 and 24 h using Eclipse Ts2-FL microscope and DS-Fi3 Camera (Nikon). Cell migration efficiency (%) was calculated by measuring the cell migration area (0–24 h) using the ImageJ software program (NIH).
9
Cell Viability Evaluation: MTT and Crystal Violet
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Cell viability was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and by crystal violet staining as previously described (76 (link), 77 (link)). Briefly, the MTT assay was conducted using the CellTiter 96 nonradioactive cell proliferation assay (MTT) (Promega; Madison, WI, USA). Cells were plated in a 96‐well plate at a concentration of 3 × 106 cells/mL in 100 μL of medium. The MTT solution (15 μL) was added to each well, and after 2 to 4 h, the reaction was stopped by the addition of 100 μL of 10% SDS. Absorbance values were acquired using an Infinite 200 PRO (Tecan, Männedorf, Switzerland) multimode plate reader at a 570-nm wavelength.
For crystal violet staining, cells were fixed in 6% formaldehyde and incubated with 0.1% crystal violet for 15 min. Unbound staining was then washed with H2O, and cells were imaged using a Nikon Eclipse Ts2-FL microscope.
For crystal violet staining, cells were fixed in 6% formaldehyde and incubated with 0.1% crystal violet for 15 min. Unbound staining was then washed with H2O, and cells were imaged using a Nikon Eclipse Ts2-FL microscope.
10
Conditioned Media Effects on Cell Migration
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We prepared different conditioned media from cocultures of HSC-2 and stromal cells (HDF, PDS1, or PDS2) or HSC-2 cells alone. To prepare the conditioned media, tumor cells and stromal cells were plated in a ratio of 1:3 and cultured for 24 hours. The media were replaced with fresh serum-free α-MEM (Invitrogen). Cells were further cultured for 48 hours, and then conditioned media were collected and filtered with a 0.2 μm–pore filter (Steradisc 25, Kurabo). For the migration assay, isolated BM cells were seeded in a 6-well plate and grown for 24 hours to approximately 90% confluence. The medium was removed, and cell monolayers were wounded by manual scraping with a sterile P200 micropipette tip. Debris was washed off with PBS 3 times. Cells were then cultured in different conditioned media with 10% FBS (Invitrogen) at 37°C in a humidified atmosphere with 5% CO2. Images were captured immediately and subsequently at 3, 6, and 9 hours using an Eclipse Ts2-FL microscope (Nikon). The percentage of the area closed was calculated using ImageJ (NIH, v1.52a). Independent experiments were repeated 3 times.
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