Human melanocytes were cultured under 3D non-adhesive conditions with and without the addition of the drug, to obtain three-dimensional spheroids and to study the effect of fucoxanthin on melanin accumulation by melanocytes under these conditions, which are closer to the native tissue compared to monolayer culture. Agarose plates were obtained by polymerizing 2% agarose type I (A6013, Sigma) in special plastic forms (12–256, 3D Petri Dishes, Microtissue) and were then placed in 12-well culture plates. Cell suspension was obtained from the monolayer melanocyte culture at passage 4, resuspended in the full growth medium, and placed in agarose plates at a concentration of 1 × 103 cells per micro-well. 50μM of fucoxanthin was added to experimental spheroids. In the control group, the same volume of sodium chloride (NaCl) buffer was added to the growth medium. The dynamics of spheroids' formation in agarose plates were monitored by the Cell-IQ live time-lapse system (CM Technologies, Finland) with photo registration every 20 min. The resulting spheroids were collected on Days 1, 3, and 7 for melanin concentration measurements and real-time PCR, and at Days 3 and 7 for immunocytochemical analysis.
A6013
Manufactured by Merck Group
Sourced in United States
The A6013 is a laboratory instrument manufactured by Merck Group. It is designed for the analysis and measurement of samples in a research or laboratory setting. The core function of the A6013 is to provide accurate and reliable data for scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
C6158, by Merck Group (1 mentions)
Vt1000, by Leica (1 mentions)
F8858, by Thermo Fisher Scientific (1 mentions)
C16509, by Thermo Fisher Scientific (1 mentions)
P35g 1.5 14 c, by MatTek (1 mentions)
Cls3527, by Corning (1 mentions)
Cc050, by Merck Group (1 mentions)
R4130, by Merck Group (1 mentions)
6 protocols using a6013
1
Melanocyte Spheroids for Fucoxanthin's Effects
2
Quantifying Infectious Virus Titer via Plaque Assay
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Plaque assays to measure infectious virus counts were performed as described before [27 (link)]. Briefly, Vero E6 cells were seeded in 6-well cell culture dishes to reach complete confluency the next day. Cells were washed once with 2 mL warm PBS and incubated with dilutions of cell culture supernatants in 200 μL complete DMEM for 1 h at 37 °C. The virus inoculum was then removed, and cells were overlaid with DMEM containing 2% FBS and 0.8% agarose (#A6013, Sigma-Aldrich, St. Louis, MO, USA). After 72 h incubation, cells were fixed with 4% formalin, and plaques were visualized via crystal violet staining (#C6158, Sigma-Aldrich, St. Louis, MO, USA).
3
Axon Tracing in E17.5 Mouse Brains
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The E17.5 brains were collected and fixed as described above. After replacing the fixative with fresh 4% PFA in 0.1 M PB, the samples were kept at 4°C until dye tracing. Samples stored less than 2 months were used. For corticothalamic or contralateral commissural axon tracing, a small Neuro-DiI (Promokine, PK-CA707-60016) crystal was placed in the primary somatosensory cortex (S1). For thalamocortical axon tracing, the crystal was placed in the thalamus. To accelerate diffusion, the samples were incubated at 37°C. The samples were then embedded in 4% agarose (Sigma, A6013), and sections at 100 μm thickness with a vibratome (Leica, VT1000S). Free-floating sections were counterstained with DAPI and then mounted on a glass slide with glycerol for examination.
4
Fluorescent Bead Sample Preparation
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To create a mixed fluorescent bead sample, we embedded three types of fluorescent beads (F8858, C16509, F8831, Thermo Fisher) into agarose gels. First, we diluted the bead suspensions and sonicated them. Then, we pipetted 10uL of the 4um bead suspension of the bead suspension , and of the bead suspension into of PBS (10010023, Thermo Fisher) for each type of bead. We mixed of the diluted bead solution, of the diluted bead solution, and of the diluted bead solution to create the final mixed beads solution, which contained approximately , and .
We prepared the agarose gel by making a [weight/volume] solution of low melting point agarose (A6013, Sigma-Aldrich) in PBS, heating it until it completely dissolved, and cooling it down to approximately . We added of the mixed beads solution to of the agarose solution. After sonication, we added a drop of the mixture onto a glass bottom dish (P35G-1.5-14-C, Mattek) and allowed it to solidify for a few minutes. Finally, we imaged the thick gel, which contained immobilized fluorescent beads, using the methods described in the main text.
We prepared the agarose gel by making a [weight/volume] solution of low melting point agarose (A6013, Sigma-Aldrich) in PBS, heating it until it completely dissolved, and cooling it down to approximately . We added of the mixed beads solution to of the agarose solution. After sonication, we added a drop of the mixture onto a glass bottom dish (P35G-1.5-14-C, Mattek) and allowed it to solidify for a few minutes. Finally, we imaged the thick gel, which contained immobilized fluorescent beads, using the methods described in the main text.
5
Agarose Gel Pillar Culture System
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To make the agarose gel pillars (1.5% w/v), agarose powder (A6013, Sigma Aldrich) was dissolved in distilled water and autoclaved for 30 min. A 5 mm thick layer of agarose gel was formed after pouring and cooling of 30 ml of the agarose liquid solution into a 9.4 cm glass Petri dish. The gel was then perforated using a 10 mm diameter punch biopsy (69036-100, Harris Uni-core) into multiple cylinder-shaped pillars. Pillars were then placed in the wells of 24-well culture plates (CLS3527, Corning Star). They were entirely covered with culture medium and left in the incubator overnight. The next day, at the start of the experiments, the old medium was replaced by fresh medium. Each agarose pillar was then loaded with one testicular tissue fragment. Tissue inserted in this system were not in direct contact with the culture medium but with the agarose surface (Figure 1B ).
6
3D Tumor Spheroid Invasion Assay
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We seeded 1000 cancer cells/spheroids into a 35 or 81-well agarose tube (A6013, Sigma-Aldrich, USA), which was generated using a 3D Petri dish, a spheroid formation device developed by Microtissues (Microtissues® Inc., RI, USA). After seeding the cells for 1 min, add 1 mL of cell culture medium for a 35-well plate or 2 mL for an 81-well plate. Incubate at 37 °C with 5 % CO2 to promote the formation of tumor spheres. Macrophages and cancer cells were added to the cancer spheroids in a ratio of 5:1. They were then co-cultured in RPMI (R4130, Sigma-Aldrich, USA) supplemented with 10 % fetal bovine serum and 100 units/mL of penicillin/streptomycin. After a 2-day co-culturing period, the invasion into type I collagen (CC050, Sigma-Aldrich, USA) was initiated.
To begin, neutralize the collagen to achieve a pH value of 7.0–8.0. Then, embed the co-culture into 35-well agarose gel tubes. Following a 4-minute incubation, invert the agarose tube containing collagen embedding co-culture and continue incubating for 1 hour. Next, invert the tube and add RPMI medium containing 5 % FBS and 1 % Pen/Strep. Perform invasion assays for 2 days and acquire images using an inverted microscope [56] (link).
To begin, neutralize the collagen to achieve a pH value of 7.0–8.0. Then, embed the co-culture into 35-well agarose gel tubes. Following a 4-minute incubation, invert the agarose tube containing collagen embedding co-culture and continue incubating for 1 hour. Next, invert the tube and add RPMI medium containing 5 % FBS and 1 % Pen/Strep. Perform invasion assays for 2 days and acquire images using an inverted microscope [56] (link).
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