MDA-MB-231 or 4T1 cells were cultured in 8-well chambers (BD Biosciences) and fixed with 4% paraformaldehyde/PBS for 15 min at room temperature. For 3D cultures cells were seeded (4 × 103 cells/well) in quadruplicate onto Matrigel (R&D, cultrex) in 8-well culture slides as described by Farias et al. [4] (link). Confocal microscopy was performed using a Leica SP5 confocal microscope. Colony morphology was determined by phase contrast microscopy (Nikon TS100 inverted phase contrast microscope) and later captured on ECLIPSE E600 microscope (Nikon Corporation, Tokyo, Japan). Cells were stained with respected antibodies as listed in the figures and the dilutions and antibodies used are listed in the supplementary table (Supplementary table 3 SI Table 3).
Ts100 inverted phase contrast microscope
Manufactured by Nikon
Sourced in Japan
The TS100 inverted phase contrast microscope is a laboratory instrument designed for observation and analysis of specimens. It features phase contrast optics, which enhances the visibility of transparent samples. The microscope provides a stable and reliable platform for various applications in fields such as cell biology, tissue culture, and materials science.
Automatically generated - may contain errors
Lab products found in correlation
8 well chambers, by BD (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Matrigel matrix growth factor reduced, by BD (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
5 protocols using ts100 inverted phase contrast microscope
1
3D Cell Culture and Microscopic Imaging
2
3D Cell Culture Assay for Breast Cancer
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3D cell culture assays were performed in 24-well plates (Thermo Fisher Scientific, Inc.) with 400 µl/well of Matrigel™ Matrix Growth Factor Reduced (BD Biosciences). Cells were suspended in complete medium supplemented with 2% Matrigel™, plated at a density of 1 × 103 for 4T1 and 3 × 103 cells/well for MDA-MB-231 and MDA-MB-157 cells respectively and incubated at 37°C for 5–12 days. A fresh layer of complete medium supplemented with Matrigel™ was added following 2 days of incubation, and the medium was replaced every 48 h with fresh DMEM medium supplemented with 2% Matrigel. Colonies formed after 5–7 days were monitored daily and images were captured under phase contrast microscope (Nikon TS100 inverted phase contrast microscope).
3
Wound-Healing Assay for Cell Migration
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Cell migration was evaluated by wound-healing assay. Briefly, cells with different treatments were seeded into 6-well plates and cultured to confluence. An artificial wound was created using a sterile 200-μL tip, and floating cells were washed away with saline buffer. Cells were cultured with serum-free medium for additional 48 h. The images of wounds were captured using an TS100 inverted phase contrast microscope (Nikon) rat 0 h and 48 h, and the cell migration ability was calculated.
4
Investigating Microtissue Dynamics via Re-plating
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Re-plating of microtissues is our newly proposed method to investigate the physical changes in the microtissues on a culture dish and examine the viability of the microtissues. The microtissues of HaCaT and ORL-48 were extracted from the calcium alginate microcapsules and re-plated in a petri dish. The released 3D microtissues from the calcium alginate microcapsules were washed three times in HBSS and then transferred to two independent petri dishes with a diameter of 35 mm. Two mL of DMEM was then added into the petri dish. The physical changes in the microtissues following the removal of the alginate shell were monitored every 24 h up to 72 h. The effects of re-plating 3D microtissues were observed and captured using a TS100 inverted phase contrast microscope (Nikon, Tokyo, Japan) that was linked to a Go-5 CCD digital camera (QImaging, Surrey, UK).
5
Microscopy Analysis of Cell Differentiation
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Cells were fixed in 4 % paraformaldehyde for 10 minutes before and after differentiation (D7), permeabilized with 0.5 percent (v/v) Triton X-100 in PBS for 5 minutes, then blocked with 5% (v/v) normal goat serum in PBS. VECTASHIELD mounting media (Vector Laboratories, Peterborough, UK) was used to mount coverslips on cover slides. Images of multinucleated cells (n=150) were captured using an NIKON Inverted Research Eclipse TiE Laser Scanning Confocal/NSIM microscope with a Galvano mode NIKON A1 RMP detector and Plan Apochromat VC 100x oil DIC N2 / 1.40 135 NA /1.515 RI objective and an extra 4x digital zoom. For the green channel, a multi-line Argon-Krypton laser (ex-457/488/561 nm) operating at 3% was used. A 0.25 m step size was retained for Z stacks. By using the 10X objective of a Nikon TS100 Inverted Phase Contrast Microscope, 200 cells (containing mono and multinucleated cells) were examined to studying the differentiation potentiality of each sample (WT LA, Mutant LAs).
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