T2904

Cells were plated onto laminin-coated (Sigma, L2020, 3.3 μg/mL) coverslips which had been treated with the self-assembled monolayer, N-1(3-[trimethoxysilyl]propyl)-diethylenetriamine (DETA, United Chemical Technologies Inc., Bristol, PA, T2910KG). Briefly, plasma-cleaned glass coverslips (Thomas Scientific 1217N81, 18 mm, no. 1.5) were silanized with a 0.1% (v/v) mixture of DETA in freshly distilled toluene (Fisher T2904) as described previously⁷³ . The DETA-treated glass was heated to just below the boiling point of toluene (70°C), rinsed with toluene, reheated to 70°C, and then dried in an oven overnight at (100–105°C). Surfaces were validated via contact angle measurement and X-ray photoelectron spectroscopy as described previously^{74 (link)}. Laminin was added to DETA-treated glass overnight at 4°C. DETA has previously been used for long-term culture of multiple cell types, including neurons^{72 (link),74 (link)–77} and enhances the adsorption of laminin onto the culture surfaces (see Supplemental Figure 1).

Glass coverslips (Thomas Scientific 1217N81, 18 mm, no. 1.5) were cleaned using an oxygen plasma cleaner (Harrick PDC-32G) for 10 min at 100 mTorr. The N-1(3-[trimethoxysilyl]propyl)-diethylenetriamine (DETA, United Chemical Technologies Inc., Bristol, PA, T2910KG) self-assembled monolayers were produced by reaction of plasma-cleaned glass with a 0.1% (v/v) mixture of the DETA in freshly distilled toluene (Fisher T2904) as described by Ravenscroft et al. (1998).¹² The DETA-coated coverslips were heated to just below the boiling point of toluene (70°C), thoroughly rinsed with toluene, reheated to 70°C, and then oven dried. Surfaces were validated prior to use in cell culture using contact angle measurement and X-ray photoelectron spectroscopy as described previously.^{19 (link)}

Glass coverslips (6661F52, 22 × 22 mm No.1; Thomas Scientific, Swedesboro, NJ, USA) were cleaned using HCl/methanol (1:1) for a minimum of 2 h, rinsed with water, soaked in concentrated H₂SO₄ for at least 2 h and rinsed with water. The coverslips were then boiled in nanopure water and oven dried. The trimethoxysilylpropyldiethylenetri-amine (DETA, T2910KG; United Chemical Technologies Inc., Bristol, PA, USA) film was formed by the reaction of the cleaned surfaces with a 0.1% (v/v) mixture of the organosilane in freshly distilled toluene (T2904; Fisher, Suwanne, GA, USA). The DETA coated coverslips were heated to ~80 °C, cooled to room temperature (RT), rinsed with toluene, reheated to approximately 80 °C, and then cured for at least 2 h at 110 °C. Surfaces were characterized by contact angle and X-ray photo-electron spectroscopy as previously described [23 (link)–25 (link)].