Fitc labeled dextran

Unlabeled poly-l-lysine
(molecular weight (M_W) 15–30 kDa)
and fluorescently labeled FITC-poly-l-lysine (M_W 15–-30 kDa) were purchased from Sigma-Aldrich.
Individual nucleotides (ATP and GTP) were purchased from Thermo Scientific.
Cy5-labeled polylysine (M_W 25 kDa) was
purchased from Nanocs Inc. Actin (rabbit skeletal muscle α actin),
fluorescently labeled ATT0 532-actin (rabbit skeletal muscle α
actin), and ATT0 594-actin were purchased from HYPERMOL in the form
of lyophilized powders. The composition of the reconstitution buffer
to dissolve actin monomers was 2 mM Tris (pH 8.0), 0.4 mM ATP, 0.1
mM CaCl₂, and 0.01 mM dithiothereitol. The end composition
of the actin polymerization buffer was 0.01 M imidazole pH 7.4, 0.1
M KCl, and 2 mM MgCl₂. Fluorescently labeled ATTO-594-phalloidin
was purchased from HYPERMOL (Cat. No. C8815-01). For permeability
experiments, we used various FITC-labeled dextran solutions: M_W 3–5 kDa (Sigma, Cat. No. FD4; mol FITC/mol
glucose = 0.001–0.02), FITC-labeled dextran M_W 20
kDa (Sigma, Cat. No. FD20S; mol FITC/mol glucose = 0.003-0.02), FITC-labeled
dextran M_W 70 kDa (Sigma, Cat. No. 46945;
mol FITC/mol glucose = 0.004), and FITC-labeled dextran M_W 150 kDa (Sigma, Cat. No. 46946; mol FITC/mol glucose
= 0.004) to actinosome. Polyvinyl alcohol (PVA), molecular weight
30,000–70,000, 87–90% hydrolyzed, was purchased from
Sigma-Aldrich.

We designed an additional degradation model that better captured the complexity of the basement membrane, based on a tissue penetration model described by Andrian et al. (85 (link)). Matrigel (Corning) was diluted 1:3 in cold PBS, and 100 μL were added to 0.4 μm polycarbonate trans-well plate inserts (VWR). The Transwell plates were placed in a temperature of 4°C for 30 min to let the Matrigel settle, and they were then moved to an anaerobic chamber to gel at 37°C for 24 h. The next day, the Matrigel was rehydrated in 100 μL of sterile reduced PBS for 1 h at 37°C. In the meantime, a 10 mg/mL stock of 40 kDa FITC-labeled dextran (Millipore Sigma) was prepared and later diluted in either media or supernatant at a final concentration of 0.5 mg/mL. 150 μL of the supernatant containing FITC-labeled dextran was pipetted on top of the Matrigel, and 300 μL of PBS were added to the lower chamber. The Transwell plates were then incubated anaerobically for 24 h at 37°C. Fluorescence in the bottom chamber was measured to assess the permeability of the Matrigel layer. Because dextran is a carbohydrate that could be digested by gut bacteria, the percentage of dextran that successfully traversed the membrane was calculated in comparison to the fluorescence levels in the leftover FITC-dextran and supernatant solution after the same anaerobic incubation at 37°C.