Tetramethylethylenediamine

Rheology measurements were made with an AR-G2 stress controlled rheometer (TA Instruments). Alginate gels were deposited directly onto the surface plate of the rheometer immediately after mixing with the crosslinker. A 20 mm plate was immediately brought down, forming a 20 mm disk of gel with an average thickness of ~1.8 mm. The mechanical properties were then measured over time until the storage modulus reached an equilibrium value. The storage modulus at 0.5% strain and at 1 Hz was recorded periodically for 45 minutes. Then, a strain sweep was performed to confirm this value was within the linear elastic regime, followed by a frequency sweep. No prestress was applied to the gels for these measurements.
The initial elastic moduli and stress relaxation properties of alginate gels were measured from compression tests of the gel disks (15 mm in diameter, 2 mm thick, equilibrated in DMEM for 24 hr) using a previously published method^{4 (link),35 (link)}. The gel disks were compressed to 15% strain with a deformation rate of 1 mm/min using an Instron 3342 single column apparatus. Within 15% compression, the stress vs. strain relations of the gels are almost linear, and the slope of the stress-strain curves (first 5–10% of strain) gives the initial elastic modulus. Subsequently, the strain was held constant, while the load was recorded as a function of time. Compression and stress relaxation measurements of polyacrylamide hydrogels and biological tissues were performed using the same procedure. Polyacrylamide hydrogels were formed following previously established protocols^{28 (link)}. In brief, 0.2 g of acrylamide and 0.02 g of bis-acrylamide were dissolved in 2 mL of water. Then 60 μL of 137 mg/mL ammonium persulfate and 60 μL of 70 mg/mL tetramethylethylenediamine (TEMED) were added into above mixture. The solution was mixed and allowed to gel for 6 hours. The hydrogel was then equilibrated in PBS for 24 hours before mechanical testing. We note that some stress relaxation of the covalently crosslinked hydrogels is observed at longer timescales, but this was previously found to arise from water leaving the hydrogel under bulk compression^{35 (link)}. Sprague Dawley Rats (male, 7 weeks of age, Charles River Lab) were euthanized in compliance with National Institutes of Health and institutional guidelines. Brain, liver, and adipose were collected immediately after euthanization and tested with Instron 3342 single column apparatus. Bone marrow from multiple rat femurs and tibias were collected fresh after euthanization and allowed to coagulate for 1 hr before compression testing. A fracture hematoma from human patient was retrieved from the bone fracture site at the moment of bone stabilization surgery. The surgery took place 7 days after occurrence of the fracture, when the surrounding soft tissue trauma around the fracture gap was sufficiently stabilized. The complete hematoma was collected and processed for mechanical testing within 1h after surgery. The same procedure of compression and relaxation measurements was performed as with the rat samples but on a Bose TestBench LM1 system using a 250g load cell. Care was taken to not test samples that contained bone chips. Fracture hematoma collection was approved by the Institutional Review Board of the Charité University Hospital Berlin, where the collection and testing were performed, and the participant gave written informed consent. Stress relaxation tests that were noisy were smoothed with a Savitzky-Golay filter in Igor Pro (Wavemetrics) with a 4s window.

SU-8 microposts were fabricated on mechanical grade silicon wafers by standard soft lithography methods. SU-8 2025 photoresist (Y111069, MicroChem) was spun to a thickness of (typically) 30 µm according to manufacturer guidelines and exposed to 365 nm UV light at ~40 mW/cm² for 12 s under a mylar mask printed with 20 µm circular features at 20,000 dpi (CAD/Art Services). The features were arranged in a square configuration with a pitch of 500 µm in the direction of separations and 190 µm in the transverse direction (a pitch of 700 µm yielded separation lengths sufficient for NEST^β to fully enter the scWestern gel). 2×8 blocks of 14×30 features (6,720 total) were spaced 9 mm apart to match the dimensions of a 2×8 well microarray hybridization cassette (AHC1X16, ArrayIt). 1 mm-thick rails spanning the length of the micropost array at a spacing of 24 mm were also patterned to support glass substrates at the height of the microposts. Uniformity of features was verified by optical profilometry after exposure and development using SU-8 developer solution (Y020100, MicroChem). The measured feature heights and diameters within a micropost block were 30.30 ± 0.15 µm (± S.D., n = 4 microposts) and 20.52 ± 0.68 µm (± S.D., n = 4 microposts) for respective nominal dimensions of 30 µm and 20 µm. Between-block CV’s in the height and diameter measurements for blocks spaced across the full length of the array were 1.1% and 5.2%, respectively (n = 3 microposts). Wafers were silanized by vapor-deposition of 2 ml of the hydrophobic silane dichlorodimethylsilane (DCDMS, 440272, Sigma-Aldrich) for 1 hour in vacuo, washed thoroughly with deionized (DI) water, and dried under a nitrogen stream immediately prior to use. Silanized wafers were robust to reuse after rinsing with DI water in excess of 20 times with moderate delamination of micropost structures.
Plain glass microscope slides (48300-047, VWR) were silanized to establish a self-assembled surface monolayer of methacrylate functional groups according to standard protocols⁴⁴ . Silanized slides were placed face-down onto micropost wafers and manually aligned to the SU-8 rail and micropost features. Gel precursor solutions were 8%T (wt/vol total acrylamides), 2.7%C (wt/wt of the crosslinker N,N’-methylenebisacrylamide) from a 30%T, 2.7%C stock (A3699, Sigma-Aldrich); 3 mM BPMAC from a 100 mM stock in DMSO, 0.1% SDS (161-0301, BioRad), 0.1% Triton X-100 (BP151, Fisher), 0.0006% riboflavin 5’ phosphate (F1392, Sigma-Aldrich), 0.015% ammonium persulfate (APS, A3678, Sigma-Aldrich), and 0.05% tetramethylethylenediamine (TEMED, T9281, Sigma-Aldrich) in 75 mM tris buffer titrated with HCl to a pH of 8.8. For confocal imaging of cells in rhodamine-tagged scWestern gels, the precursor included the fluorescent monomer methacryloxyethyl thiocarbamoyl rhodamine B (23591, Polysciences) at 3 µM from a 100 µM stock in DMSO. The precursor mixture was sonicated and degassed (Aquasonic 50D, VWR) for 1 min in vacuo immediately prior to the addition of detergents (SDS, Triton) and polymerization initiators (riboflavin, APS, TEMED). The precursor was then injected into the gap between the glass slide and silicon wafer using a standard 200 µl pipet. After allowing ~30 s for precursor to wick through the gap, the slide was exposed to blue light for 7.5 min at 470 lux (advanced light meter, 840022, Sper Scientific) from a collimated 470 nm LED (M470L2-C1, Thor Labs) mounted at a 45° angle above the slide. Polymerization was allowed to continue for an additional 11 min. Gel-fabricated glass slides were wetted at their edges using 2 ml of phosphate-buffered saline (PBS), pH 7.4 (21-040, Corning) and carefully levered from wafers using a razor blade. Fabricated slides could be stored at 4°C in PBS for up to 2 weeks before use without loss of protein separation or photocapture properties.