A total of 200nm yellow-green, fluorescent beads (ThermoFisher Scientific F8811) were diluted to a ratio of 1:200 in 2.0% agarose. Then, 0.2 grams of agarose powder (Bio-Rad, Certified Molecular Biology Agarose 1613101) were added to 10 milliliters of DI (Deionization) water to make the mass concentration 2.0%. Next, the agarose solution was heated in a microwave for intervals of 45 s. This process was continued until the agarose became a gel mixture. After 1–2 min passed for the agarose gel to cool down to a safe handling temperature, the agarose gel was ready to hold the beads in place for imaging analysis. In total, 2.0 ml of beads were added to 400 ml of agarose to achieve the 1:200 ratio. After drying completely, the Petri dish was then placed on the stage of the TPFM-AO system and DI water was added for imaging with the water dipping objective.
F8811
Manufactured by Thermo Fisher Scientific
The F8811 is a high-precision laboratory balance designed for accurate weighing in scientific and research applications. It features a robust construction, intuitive user interface, and reliable performance to meet the demands of a wide range of laboratory environments.
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Lab products found in correlation
Uapon, by Olympus (1 mentions)
Flash 4.0 v3, by Hamamatsu Photonics (1 mentions)
Fluospheres sulfate microspheres f8848, by Thermo Fisher Scientific (1 mentions)
Pc02n, by Bangs Laboratories (1 mentions)
Transferrin, by Solarbio (1 mentions)
Fluorescent beads, by Thermo Fisher Scientific (1 mentions)
Il 17a, by R&D Systems (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Sulfo sanpah, by Thermo Fisher Scientific (1 mentions)
Insulin, by Solarbio (1 mentions)
Ixon ultra emccd camera, by Oxford Instruments (1 mentions)
Csu w1 confocal unit, by Yokogawa (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
N n methylenebisacrylamide bis, by Merck Group (1 mentions)
Methylene blue, by Maclin Biochemical Technology (1 mentions)
Pegda, by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
Uplsapo 60xw 1.2 na, by Olympus (1 mentions)
12 protocols using f8811
1
Fluorescent Bead Suspension in Agarose Gel
2
Structured Illumination Microscopy of Cells
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A commercial structured illumination microscope (HIS-SIM, Guangzhou Computational Super-resolution Biotech) was used to acquire the cell images, which was based on a commercial inverted fluorescence microscope (IX83, Olympus), equipped with two objectives (×100/1.7 HI oil, APON, Olympus; ×100/1.49 oil, UAPON, Olympus), four excitation wavelength lasers (405 nm, 488 nm, 561 nm, and 640 nm), a multiband dichroic mirror (ZT405/488/561/640-phase R, Chroma) and an sCMOS camera (Flash 4.0 V3, Hamamatsu)10 (link). HIS-SIM is controlled by its own software Imager (v1.1.23d). The raw image obtained has a pixel size of 65 nm. Generally, the exposure time of each frame of the raw image is 5 ms or 10 ms. The OTF used for reconstruction was prepared from actual fluorescent bead images (40 nm in diameter, ThermoFisher, F8771). And the diameter of the fluorescent microbeads used to compare the reconstruction effect of different exposure times, i.e., different signal-to-noise ratios, was 200 nm (ThermoFisher, F8811). In calcium imaging experiments, we collected the first 3–5 s as the basic fluorescence levels before adding ATP solutions to stimulate cells, and set the raw image exposure for 10 ms and imaging without interval for 2 min.
3
Fluorescent Bead Assay for Cell Motility
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Fluorescent polystyrene beads 0.2 μm in size (FluoSpheres sulfate microspheres F8848, carboxylate-modified microspheres F8811, amine-modified microsphere F8764; Thermo Fisher) were diluted 300 times to 0.02% (wt/vol) in BG11 and used for the bead assay, as previously described (7) (link). A coverslip was coated with 0.2% (vol/vol) collodion in isoamyl acetate and air-dried before use. The cell culture was poured into a tunnel chamber assembled by taping a coverslip. After incubation at 45 °C for 2 min on the microscope stage, the cells were subjected to vertical illumination from blue-light LED through a dark-field condenser at a fluence rate of 200 μmol m -2 s -1 . After illumination for 2 min, fluorescent beads were added to the sample chamber, and their movement was visualized by blue light illumination at 0.1-s intervals. Lateral illumination from green-light LEDs was applied for 2 min before adding the beads if needed.
4
Fluorescent Microsphere Dilution
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Carboxylate-modified fluorescent microspheres, diameter 200 nm, with 488 nm fluorescence (F8811) were purchased from Invitrogen with a concentration of 4.5 × 1012 beads per ml. The beads were diluted in DPBS to a concentration of 4.5 × 107 beads per ml.
5
Polystyrene Nanoparticle Characterization
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Polystyrene NPs were used in all
experiments (Table1 ). The diameter provided
by the supplier is used to identify the NP: 93 nm carboxylate-modified
(Bang’s Laboratories, FC02F), 87 nm amine-modified (Invitrogen,
C29029), 200 nm carboxylate-modified (Invitrogen, F8811), 200 nm amine-modified
(Invitrogen, F8764), 60 nm carboxylate-modified (Bang’s Laboratories,
PC02N), and 58 nm amine-modified (Bang’s Laboratories, PA02N).
experiments (Table
by the supplier is used to identify the NP: 93 nm carboxylate-modified
(Bang’s Laboratories, FC02F), 87 nm amine-modified (Invitrogen,
C29029), 200 nm carboxylate-modified (Invitrogen, F8811), 200 nm amine-modified
(Invitrogen, F8764), 60 nm carboxylate-modified (Bang’s Laboratories,
PC02N), and 58 nm amine-modified (Bang’s Laboratories, PA02N).
6
Saponins Modulate Cytokine-Mediated Cellular Functions
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Saponins of Dioscorea nipponicae was purchased from Hangzhou Dawen Biotec Co., Ltd. (Hangzhou, Zhejiang, China), IL-17A was purchased from R&D Systems (Minneapolis, MN). Collagen type I, RGD peptide (amino acid sequence Arg-Gly-Asp) and 3-aminopropyltrimemethoxysilane were obtained from Sigma-Aldrich (St. Louis, MO). Sulfo-SANPAH was purchased from Pierce Biotechnology, Inc. (Rockford, IL). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Insulin and transferrin were purchased from Solarbio (Beijing, China). Fluorescent beads of 0.2 μm diameter (F8811) were purchased from Invitrogen Life Technologies (Carlsbad, CA).
7
Nodal Flow Analysis via Confocal Microscopy
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Nodal flow was observed by multipoint scanning confocal microscopy. Particle image velocimetry (PIV) analysis was performed as described previously18 (link). Recovered embryos were first cultured under 5% CO2 for 30 min at 37 °C in DMEM supplemented with 75% rat serum. The region containing the node was then excised, and the node cavity was filled with DMEM supplemented with 10% FBS and 0.2 µm-diameter fluorescent microbeads (Invitrogen, F8811). The motion of the beads was monitored for 10 s in planes of +5 and +10 µm relative to the cavity (21 frames per second) with the use of a CSU-W1 confocal unit (Yokogawa) and an iXon-Ultra EMCCD camera (Andor Technology) connected to an IX83 microscope (Olympus) fitted with a ×60 objective lens. Time-series images for PIV analysis were captured at a resolution of 512 by 512 pixels and were processed with interrogation windows of 16 by 16 pixels with 50% overlap, corresponding to a spatial resolution of 4.3 by 4.3 mm. The time-averaged velocity distributions were calculated for 10-s intervals.
8
Multifunctional Hydrogel Synthesis Protocol
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PEGDA (average Mn = 700), N,N′-methylenebis (acrylamide) (BIS), and photoinitiator 2-hydroxy-2-methylphenylpropanone (HMPP) were all procured from Sigma-Aldrich. Fluorescent polystyrene (PS) nanoparticles (F8805 and F8811) were procured from Invitrogen. GO solution (2 mg/ml) was bought from XF NANO Co. Ltd. NIPAM (97%) and methylene blue (MB) were bought from Macklin. Photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) was derived from Aladdin. Magnetic nanoparticles were synthesized by a hydrothermal method. Silicone oil (50 cSt) was purchased from Shin-Etsu Chemical Co. Ltd.
9
Nodal Flow Visualization Using PIV
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Nodal flow was observed with an IX83/CSU-W1 microscope equipped with UPLanSAPO 60× Oil lens and EMCCD camera as mentioned above, and particle image velocimetry (PIV) analysis was performed as described previously (14 (link)). The excised node region was treated with 75% rat serum/DMEM containing 0.2-μm fluorescence beads (505/515; F8811, Invitrogen), and the nodal cavity was filled with the medium containing the beads. The motion of the beads was monitored in planes of ~10 μm above the surface of node pit cells.
10
Fluorescent Bead Dilution Protocol
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Carboxylate-modified fluorescent microspheres, diameter 200 nm, with 488 nm fluorescence (F8811) were purchased from Invitrogen with a concentration of 4.5 × 1012 beads per ml. The beads were diluted 1:100 and 1:1,000 in DPBS.
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