Aclar fluoropolymer film

5 × 10⁵ 3T3/OKT3-1, OKT3-2, OKT3-3, OKT3-CD43, OKT3-7, or OKT3-CD44 cells were grown overnight on an Aclar fluoropolymer film (Electron Microscopy Sciences, Hatfield, PA, USA) in a 24-well plate and incubated with 5 × 10⁵ Jurkat T cells for 2 h at 37°C. After washing with PBS, the cells were fixed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 2 h, and then post-fixed in osmium tetroxide for 1 h. Samples were washed with 0.1 M cacodylate buffer three times (15 min/wash) and then dehydrated in a series of ethanol from 30 to 100%. After dehydration, the samples were placed in acetone and infiltrated with Spurr’s resin. Polymerization of Spurr’s resin was performed by heating to 60°C for 48 h. Ultrathin sections (60–90 nm) were cut on a Reichart-Jung Ultracut E microtome and then mounted on 300 mesh copper grids. The sections were stained in saturated aqueous uranyl acetate and Reynold’s lead citrate, washed with distilled water, and examined on a Hitachi H-7000 transmission electron microscope at 150,000× magnification. Analysis of intercellular distances was performed with Adobe Photoshop CS5 by dividing the conjugation area between two cells into equally spaced sections (11–20) and measuring the distance at each point. Nine to 15 individual cell conjugates were analyzed for each group.

Overnight cultures were diluted 1:10 in fresh LB medium and were added to 48-well microtiter plates containing autoclaved Aclar fluoropolymer film (Electron Microscopy Sciences, Hatfield, PA). Biofilms were grown for 48 h at 37 °C, shaking at 50 rpm, and prepared for SEM using cationic dye stabilization methods.^{57 (link),58 (link)} Briefly, Aclar membranes containing biofilm growth were washed three times in 0.2 M sodium cacodylate buffer, and submerged in primary fixative (0.15 M sodium cacodylate buffer, pH 7.4, 2% paraformaldehyde, 2% glutaraldehyde, 4% sucrose, 0.15% alcian blue 8 GX) for 22 h. Samples were washed three more times prior to a 90 minute treatment with secondary fixative (1% osmium tetroxide, 1.5% potassium ferrocyanide, 0.135M sodium cacodylate, pH 7.4). After three final washes, biofilms were chemically dehydrated in a graded ethanol series (25, 50, 70, 85, 95 [2×] and 100% [2×]) before CO₂-based critical point drying. Aclar membranes were attached to SEM specimen mounts using carbon conductive adhesive tape and sputter coated with ~ 5 nm iridium using the Leica ACE 600 magnetron-based system. Biofilms were imaged using a Hitachi S-4700 field emission SEM with an operating voltage of 2 kV.