Draq7

DNA was stained with 2µM YoYo1 Iodide (excitation/emission peaks = 458 nm/564 nm;
Invitrogen, Grand Island, NY) diluted in blocking solution for 30 minutes at room temperature
in a humidified chamber in the dark and washed in blocking solution. Alternatively, DNA was
stained with 1.2 μM DRAQ 7 (excitation/ emission peaks = 633nm/695nm; Abcam, Cambridge,
MA) diluted in blocking solution for one hour at room temperature in a humidified chamber in
the dark, transferring to a fresh drop every 20 minutes. The oocytes were then washed in blocking
solution. It should be noted that both DNA stains were used interchangeably through the experiments
as their only purpose was to visualize DNA. No measurements were taken of the fluorescence
emitted from these dyes.
Oocytes were incubated in 75% Vectashield Hardset Mounting Medium (Vector Laboratories,
Burlingame, CA) diluted in blocking solution and 100% Vectashield. Oocytes were mounted
in 5µL Vectashield on a #1 coverslip that had been dabbed on all four corners with Vaseline
containing glass beads ≥106 µm (Sigma-Aldrich, St. Louis, MO). Coverslips
were sealed to the slide with clear nail polish to prevent drying.

Depending on the pathogen, the chips were incubated immediately before infection with the applicable subsets of the following probes:
1) With E. histolytica. Before infection, both channels of the chip were filled with warm iTY (37°C) containing DRAQ7 (a far-red dye that only stains the double-stranded DNA of dead or permeabilized cells; 1/50, 6 mM final; Abcam) and, occasionally, E64 (cysteine protease inhibitor; 100 mM; Alfa Aesar). As a preliminary measure, the chip was then immediately inspected under the microscope for 10 min at 37°C. Once ready for infection, the chip was temporarily removed from the microscope, and its top channel was filled with a warm solution of iTYI containing 10⁶ trophozoites/ml together with DRAQ7 (and occasionally E64). The chip was then put back under the microscope (37°C) and connected to the microfluidic pump.
2) With S. flexneri. Before infection, both channels of the chip were filled with warm medium having one or more of Pro12A (blue membrane dye; 2 nM), SiR-Actin kit (0.2 nmol; Spirochrome), and lectin (peanut agglutinin) AF647 (1/500; stock solution: 3 mol of dye/mole; far-red dye).
Pro12A is a noncommercial membrane marker that allows labeling the membranes of the epithelial cells in blue with better specificity and less channel leaks when compared to commercial dyes. It was provided by the laboratory of A. S. Klymchenko (50 (link)).

Caco-2 cells were seeded at a density of 200.000 cells/well in flat bottom 24-wells plates. GOS or SL were added to the wells for 4 days. The supernatant was collected and quantified for secreted alkaline phosphatase by QUANTI-Blue (Invivogen, rep-qb2) as a marker of differentiation. QUANTI-Blue powder was reconstituted according to the manufacturer's instructions. 60 μl supernatant was added to 190 μl QUANTI-Blue and incubated for 3 h at 37°C. The optical density (OD) at 625 μm was measured using a FilerMAX F5 (Molecular Devices, Nederman, Germany). To count the cells, trypsin was added to the cells. The cells were spun down at 300 g for 5 min and washed twice with PBS. 150 μl Trypsin-EDTA (0.25%) (ThermoFisher, 25200) was added to detach the cell monolayer. Trypsin was inactivated by resuspending the cells in 150 μl PBS + 4% FCS + 0.02% EDTA. Dead cells were stained with DRAQ7 (Abcam; ab109202) and 50 μl of 0.975E6 beads/ml (Fluoresbrite YG Carboxylate microspheres 10 μm, 18142) were added to count cells (Figure S2A; gating strategy). Cells were acquired on a BD FACS Canto II (BD Biosciences) and analyzed using the FlowJo software V10.