Polystyrene particle suspensions were created using 4.4 μm blue-fluorescent beads (Polysciences), 9.9 μm green-fluorescent beads (ThermoFisher Scientific) and 15 μm red-fluorescent beads (Invitrogen). Each was suspended to a final length fraction of 0.1 in an equivalent density solution (1.05 g/mL) of 1x PBS, 0.1% Tween20, and iodixanol. White blood cells (buffy coat) were isolated using deterministic lateral displacement with a co-flow of buffer which has been previously published9 .
Red fluorescent beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Red fluorescent beads are spherical particles that emit red fluorescent light when excited by a light source. These beads are designed for use in various applications that require the detection of red fluorescence, such as flow cytometry, fluorescence microscopy, and immunoassays.
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Lab products found in correlation
9 protocols using red fluorescent beads
1
Particle Suspension and Cell Isolation
2
Quantifying αSyn Overexpression Effects
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A human neuroglioma H4 cell-derived cell line stably over-expressing αSyn from a tetracycline inducible promoter20 (link) was grown in serum-free X-VIVO media (Lonza Group, Basel, Switzerland). Cells were seeded in 96-well plates at 100 K cells per well. The next day, compound was added along with 5 µg/ml tetracycline to induce αSyn overexpression. Cells were cultured overnight and the next day were fed 4 μM red fluorescent beads (In Vitrogen, Carlsbad, CA) for 90 minutes at a cell-to-bead ratio of 1:10. Plates were gently washed with 100 µl/well media twice, fixed and stained with HEMA3 (Thermo Fisher, Waltham, MA). Plates were dried overnight and read on an ArrayScan (Thermo Fisher, Waltham, MA). As the HEMA3 stain absorbs light, the internalized beads are less fluorescent than the outside beads. Tet/non-tet and 484228 samples were run on each plate.
3
Cell Traction Force Measurement using Beads
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We used TFM to evaluate changes in cell contractility, as previously described [35] . In brief, to prepare gel substrates for cell traction measurements, a 0.01% suspension of 0.5-μm diameter red fluorescent beads (Invitrogen, USA) were added to the gel solution. After gel polymerization, the surface was functionalized as mentioned above. Cells were seeded and allowed to adhere and spread for two days. Phase-contrast images of the cell boundaries and the fluorescent beads at the surface of the gel were acquired.
The cells were then removed by trypsinization, and another image of the bead positions was obtained (Nikon TE-2000U, Japan).
To analyze the total force exerted by a cell on the ECM, correlation-based particle image velocimetry (MATLAB, USA) was used to compute the image registration and track bead displacements. Displacements were then applied as a boundary condition on the surface of the gel, and the root mean square (RMS) traction stresses, net contractile moment, and maximum traction field on the gel surface were calculated using the COMSOL finite element package (COMSOL Multiphysics, USA) [31] .
The cells were then removed by trypsinization, and another image of the bead positions was obtained (Nikon TE-2000U, Japan).
To analyze the total force exerted by a cell on the ECM, correlation-based particle image velocimetry (MATLAB, USA) was used to compute the image registration and track bead displacements. Displacements were then applied as a boundary condition on the surface of the gel, and the root mean square (RMS) traction stresses, net contractile moment, and maximum traction field on the gel surface were calculated using the COMSOL finite element package (COMSOL Multiphysics, USA) [31] .
4
Measuring Cell Traction Forces
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Cell traction force was determined by first measuring the hydrogel displacement field and then solving the inverse elastic problem to reconstitute the traction force map. Specifically, red fluorescent beads (200 nm, Thermo Fisher Scientific) were mixed at a density of 0.3% (v/v) with the 30 kPa PAAm pre-gel solution containing 10.4% (w/v) acrylamide and 0.264% (w/v) bis-acrylamide. After polymerization, hydrogels were activated with 0.2 mg/ml Sulfo-SANPAH and then coated with 0.1 mg/ml vimentin (62 ). hMSCs were sparsely seeded on the vimentin-coated hydrogels to minimize the cell–cell contact. A bright field image of a randomly selected cell and the corresponding fluorescent beads image was taken. The cell was then detached from the hydrogel with 0.5 N sodium hydroxide, and the reference image of the fluorescent beads was taken. The in-plane displacement field of the hydrogel substrate was calculated by particle image velocimetry. The traction force field was then constructed by solving the inverse problem of Boussinesq solution through Fourier transform traction force cytometry (63 (link), 64 ).
5
Fluorescent PDMS Substrates for Cell Culture
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Polydimethylsiloxane (PDMS; Sylgard®184, Dow Corning) was mixed at a ratio of 1:10 with 0.5 μm red fluorescent beads (ThermoFisher) and sandwiched between a Petri dish and a glass slide to gain thin and flat substrates. PDMS was degassed for 20 min, cured at 70°C for 12 h, and coated with 1μg/cm2 fibronectin (Sigma-Aldrich) for 1 hour. Cells were plated at low density (i.e., maximum of ~5 to 9 cells per 106 3 106 μm2) on substrates for 2 hours prior to hyperosmotic loading experiments.
6
Fluorescent PDMS Substrates for Cell Culture
7
Biotinylated cAMP and LPS Bead Preparation
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To make biotinylated cAMP, 50 μl of 18 mM biotin EZ-Link Sulfo-NHS-Biotin (Thermo Fisher) was incubated with 100 μl of 10 mM 6-AH-cAMP (Biolog) at 22°C for 8 h and purified by HPLC. NeutrAvidin beads with 1-μm diamter (Thermo Fisher) were washed with PB 3 times and resuspended into 1 ml PB. The beads were then incubated with biotinylated cAMP at 22°C for 2 h. The coated beads were washed 5 times with precold PB to remove excess free ligand. To make LPS-labeled beads, biotinylated bacterial LPS (E. coli O111:B4, InvivoGen) was incubated with 1 μm NeutrAvidin beads 22°C for 2 h. The coated beads were washed 5 times with precold PB to remove excess free ligand. The non-coated NeutrAvidin beads or red fluorescent beads (Thermo Fisher) were washed 5 times with PB before use.
8
Cell Traction Force Quantification
9
Cell Traction Quantification Protocol
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We followed the protocols of cell-traction quantification published previously11 (link). In brief, cells were plated on PA gels with red fluorescent beads (0.2 μm in diameter; Molecular Probes, Invitrogen) embedded on the top surface. Before and after cell trypsinization, fluorescent images were taken to compute the displacement field of the beads using digital image correlation (DIC)in a homebuilt MATLAB program. Cell tractions were then calculated from the displacement field using inverse Boussinesq mathematical model.
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