Lipids including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL). Lipid dyes BODIPY FL DHPE, triethylammonium salt, and Texas Red DHPE, triethylammonium salt were purchased from Invitrogen (Carlsbad, CA). Glass beads with diameter of 10-30 μm and Polybead carboxylate microspheres with a diameter of 1 μm were obtained from Polysciences, Inc. (Warrington, PA). D-sucrose, dextrose (D-glucose) anhydrous, phosphate buffered saline concentrate powder, and chloroform were purchased from Fisher Scientific (Hampton, NH). Poly-L-lysine 1 mM solution and bovine serum albumin were purchased from Sigma-Aldrich.
Glass beads
Manufactured by Polysciences
Sourced in Panama
Glass beads are small, spherical particles made of glass. They are commonly used in various laboratory applications as a versatile tool. Glass beads can be utilized for a range of purposes, including sample preparation, filtration, and as a reference material.
Automatically generated - may contain errors
Lab products found in correlation
Chloroform, by Thermo Fisher Scientific (1 mentions)
Polybead carboxylate microspheres, by Polysciences (1 mentions)
D sucrose, by Thermo Fisher Scientific (1 mentions)
Dextrose d glucose anhydrous, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nanodrop 3300 fluorospectrometer, by Thermo Fisher Scientific (1 mentions)
Rnasezap, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Merck Group (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Jpk bioafm nanowizard 3, by Bruker (1 mentions)
Tl cont, by Nanosensors (1 mentions)
8 protocols using glass beads
1
Lipid Bilayer Formation and Characterization
2
Measuring Polyacrylamide Gel Modulus via Bead Indentation
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The Young's moduli of polyacrylamide gels made from different formulations were measured using a bead indentation technique7 (link) based on Hertzian indentation theory27 28 (link) (Supplementary Fig. 1 ). Glass beads (Polysciences) with radii ranging from 0.03 to 5 mm were placed on 700–1,000 μm thick gels created by pipetting 300 μl of polymer solution onto an activated glass culture dish and covered with 25 mm No. 1.5 circular cover slip (Fisher 12-545-102) . The bead contact area was measured using the gravity-settled 200 nm crimson fluorospheres as a marker for the gel surface. The bead indentation depth (δ) was calculated from the bead radius (R) and the contact radius (r) according to equation (13 ).
From this indentation depth, the gel Young's modulus (E) was calculated using the Poisson ratio of the hydrogel (ν), and the buoyancy corrected bead force (f) according to equation (14 ).
For polyacrylamide gels, ν=0.3–0.5 (ν=0.3 was used here), and the glass bead density was measured to be ∼2,600 kg m−3.
From this indentation depth, the gel Young's modulus (E) was calculated using the Poisson ratio of the hydrogel (ν), and the buoyancy corrected bead force (f) according to equation (
For polyacrylamide gels, ν=0.3–0.5 (ν=0.3 was used here), and the glass bead density was measured to be ∼2,600 kg m−3.
3
Asbestos and Glass Bead Characterization
4
Quantifying Liquid Carryover in CIFF
5
RNA Capture Bead Fluidic Protocol
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The fluidic cartridge was filled with glass beads (diameter, ~30 μm;
Polysciences). Beads were suspended in 75% ethanol (Sigma) and introduced through the
inlet. The beads were retained in the RNA capture chamber due to the weir-style
physical barrier in the outlet side of the chamber (fig. S1A). Following bead
capture, excess ethanol was collected and removed. The entire device was then flushed
with cycles of RNaseZap (Life Technologies), ribonuclease (RNase)–free water
(Life Technologies), and ethanol, and dried. All fluidic flow was generated by
manually operating syringes.
Polysciences). Beads were suspended in 75% ethanol (Sigma) and introduced through the
inlet. The beads were retained in the RNA capture chamber due to the weir-style
physical barrier in the outlet side of the chamber (fig. S1A). Following bead
capture, excess ethanol was collected and removed. The entire device was then flushed
with cycles of RNaseZap (Life Technologies), ribonuclease (RNase)–free water
(Life Technologies), and ethanol, and dried. All fluidic flow was generated by
manually operating syringes.
6
Characterization of Asbestos and Glass Fibers
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Crocidolite asbestos (SPI-Chem) was used as a positive control for experiments and was previously characterized by our lab to have an average length of 13.60 ± 20.24 µm (~0.5–250 µm min/max) and an average width of 0.60 ± 0.45 µm (~0.2–5 µm min/max) (Qi et al. 2013 (link)). Due to reports of glass fibers causing tumors in rodents (Stanton et al. 1977 (link)), glass beads (Polysciences, Inc.) were used as a negative control for experiments. According to company specifications, glass beads had diameter ranges of 3–10 µm. glass beads, crocidolite, and palygorskite (Source Clays Repository) were prepared as previously described (Jube et al. 2012 (link); Qi et al. 2013 (link)). Briefly, fibers were baked for 18 h at 150ºC, added to 1× phosphate-buffered saline (PBS), and fiber bundles were disaggregated by passing through a 22-gauge needle 10 times.
7
Quantification and Normalization of Dorsal Gradient
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Quantification of Dorsal gradient and normalization method are described in detail in [8] (link). Briefly, embryos were stained for anti-Dorsal antibody (Iowa Hybridoma Bank) and a Donkey anti-mouse Alexa 647, manually sliced in cross-sections within trunk region and imaged using a LSM700 Zeiss Confocal microscope. Fluorescent intensity from the 30-most ventral nuclei was obtained using Axiovision software (Zeiss). Position of midline was estimated with a double staining for snail RNA. For nuclei diameter measurement, early-stage embryos stained with anti-Laminin (Iowa Hybridoma Bank) were mounted longitudinally with glass beads (150–210 µm size, Polysciences), to prevent flattening caused by the coverslip. Confocal slices were taken from the embryo surface to its mid-section and nuclei diameter was determined using ImageJ software. In the case of ssm and gyn mutations, some additional measurements were taken from embryos stained with DAPI nuclear dye.
8
Biomechanical Assessment of Mouse Embryonic Cartilage and Muscle
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E11.5 mouse embryos were extracted and snap-frozen in O.C.T. compound with liquid nitrogen. Those embryos were then serially cryo-sectioned by a microtome with a thickness of 20 μm. To identify nascent cartilage and muscle regions for AFM measurement, we performed immunofluorescence staining (cartilage: Sox9, muscle: MyoD) and in situ mRNA hybridization (cartilage: Col2a1, muscle: MyoD) for one-before and one-after cryo-sections, respectively. Finally, those staining results for those neighboring cryo-sections were collated with AFM results. For AFM-based tissue indentation experiment, the cryo-section was thawed and the nuclei were immediately stained with Hoechst 33342 in PBS(-). In this study, AFM system (JPK BioAFM NanoWizard 3; Bruker Nano GmbH., IX81; Olympus Co.) was used and AFM cantilevers (TL-CONT; spring constant 0.2 N/m; NANOSENSORS) were modified with glass beads (15 μm-diameter; Polysciences. Inc.). Spring constant of the AFM cantilever was calibrated with a thermal noise method. Piezo displacement speed was set to be 3 μm/s. As a result of tissue indentation, force (F) versus indentation depth (h) curves were obtained and slope (nN/μm) was evaluated for each curve by linear regression. Sample points within the force range of 750 pN≤F≤ 1000 pN were used for the evaluation. After AFM, the sample was immunostained for Sox9 and MyoD.
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