Bovine plasma fibronectin

To determine adhesion of THP-1.EV, THP-1.SIRP-ß2 and THP-1.SIRP-ß2.K202L, 7.5x10⁵ cells were added into a 6-well plate and incubated for 4 or 24 h in the presence or absence of 2.5 µg/mL PMA. After incubation, the wells were gently washed twice with PBS and stained with crystal violet (Sigma Aldrich, St. Louis, MO, USA) (1:3 dilution) for 1 h at RT. The crystal violet staining buffer was removed and wells were washed six times with PBS. Photos were taken on day 2, 4 and 7 to screen for adhesion, using an auto screen machine (AID EliSpot reader). Adhesion assay using xCELLigence, was performed as described previously (41 (link)). In short, 96-well E-plates (E-plate 96) (ACEA Bio, catalog number: 5232368001) were coated with extracellular matrix (ECM) molecule fibronectin (bovine plasma) (Sigma-Aldrich, catalog number: 341631) for 1 h at 37°C, whereafter excess coating was removed by washing twice with PBS. To block the non-specific binding of the THP-1 and HL-60 cells, the plate was coated for 1h at 37°C with 100 µl 0.1% BSA. For the experiment, 3x10⁴ cells were plated in an end volume of 150 µl complete medium and measured for 4h in real time.

To prepare the surface of PDMS for cell adhesion, dopamine hydrochloride (Sigma, ON, Canada, Product number: H8502) with a concentration of 1 mg/ml was dissolved in phosphate-buffered saline (PBS) and the pH was adjusted to 8.5 using 1 M Sodium Hydroxide (NaOH). Then, submerged monolayer chips were incubated with this solution at room temperature for 24 h as described in our previous work to create a poly-dopamine (PDA) surface (Dabaghi et al., 2021b (link)). After PDA coating, all devices were thoroughly rinsed with PBS (pH = 7.2–7.4) to remove any unattached PDA. PDA coating was followed by incubation with a solution of 25 µg/ml fibronectin bovine plasma (Sigma, ON, Canada, Product number: F4759-1 MG) and 50 µg/ml type I rat tail collagen (Advanced BioMatrix, CA, United States, product number: 5056-20 ML) together at 4°C for 24 h. Gelatin coating was performed via incubation with 1 mg/ml type A gelatin from porcine skin (Sigma, ON, Canada, Product number: 924504) at 60°C for 24 h. Collagen/fibronectin coating and gelatin coating were performed similarly in the absence of PDA coating for comparison purposes. After coating, chips were rinsed with PBS to remove unbonded molecules.

PA hydrogels were fabricated according to the protocol described by Tse and Engler (2010) (link) with some modifications: hydrogels were cast on 35 mm glass bottom dishes (D35-14-1-U, Matsunami Glass, Osaka, Japan) instead of coverslips, and deposition of NaOH was followed by immediate aspiration instead of heated evaporation. Hydrogels were then tethered with either fibronectin (FN; fibronectin bovine plasma, F1141, Sigma, St Louis, MI, USA), collagen (COL; collagen I, rat tail, A1048301, Gibco, Waltham, MA, USA) or laminin (LAM; 11243217001, Roche, Basel, Switzerland) at a 100 mg/ml concentration according the protocol reported by Tse and Engler (2010) (link). Briefly, polymerized hydrogels were covered with sulfo-SANPAH (22589, Thermo Fisher Scientific, Waltham, MA, USA), exposed to UV light for 15 min, and then washed with HEPES buffer three times for 15 min. The hydrogels are then incubated in the ECM proteins overnight at 37°C. Young's moduli of the hydrogel formulations were measured by compression testing using a Mach-1 micromechanical system (Biomomentum, Laval, Quebec, Canada) controlled by a Universal Motion Controller (Newport, Irvine, CA, USA) (Fig. S1A).
For traction force microscopy, PA hydrogels were fabricated following the same protocol above with the addition of embedding 0.1 µm diameter fluorescent beads (F8800, Thermo Fisher Scientific) at a 1:200 dilution.

NPCs were isolated from embryonic day 13.5 Ai9-tdTomato homozygous mouse brains. Cells were cultured as neurospheres at 37 °C with 5% CO₂ in NPC medium: DMEM/F12 (Gibco, CAT# 10565018) with GlutaMAX supplement, sodium pyruvate, 10 mM HEPES, nonessential amino acid (Gibco, CAT# 11140076), penicillin and streptomycin (Gibco, CAT# 10378016), 2-mercaptoethanol (Gibco, CAT# 21985023), B-27 without vitamin A (Gibco, CAT# 12587010), N2 supplement (Gibco, CAT# 17502048), and growth factors, bFGF (BioLegand, CAT# 579606) and EGF (Gibco, CAT# PHG0311) (both 20 ng/ml as final concentration). NPCs were passaged using MACS Neural Dissociation Kit (Papain, CAT# 130-092-628) following manufacturer’s protocol. bFGF and EGF were refreshed every three days and cells were passaged every 5 days. Pre-coating with a coating solution containing poly-DL-ornithine hydrobromide (Sigma-Aldrich, CAT# P8638), laminin (Sigma-Aldrich, CAT# 11243217001), fibronectin bovine plasma (Sigma-Aldrich, CAT# F4759) was required for culturing cells in 96-well plates.

Primary human brain microvascular endothelial cells (HBMECs) from ScienCell™ Research laboratories (Carlsbad, CA, USA, Catalog #1000) were used in passages 3–6 in all assays. Cells were maintained in complete Endothelial Cell Medium (ECM, ScienCell™ Research Laboratories) in a CO₂ incubator at 37 °C, 5% CO₂, and 95% humidity. Before seeding, all flasks and plates were coated in 2 μg/cm² fibronectin bovine plasma (Sigma-Aldrich, St. Louis, MO, USA) and in Mg²⁺- and Ca²⁺-free dPBS. For testing, HBMECs were grown in T75 flasks, detached using Accutase (Sigma-Aldrich), and resuspended in 1 mL glucose-depleted DMEM (Glc(-) DMEM) (Lonza, Basel, Switzerland) before being seeded in the appropriate test plates.