1. Irradiation Set-up

Note: Irradiation of Caco-2 cells was performed at the radiotherapy department of Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) S. Maugeri (Pavia, Italy) with a linear accelerator routinely used to treat different types of cancers.

1. Set photon X-ray energy to 6 MV peak. The linear accelerator operates in a pulsing regime (time between two consecutive pulses approximately of 4 ms; duration of a single pulse about 5 µs with a dose rate up to 1 × 10^-3 Gy/p).
2. Place the 6-well plates containing inserts with Caco-2 on the trajectory of X-rays on a 1.4 cm-thick Plexiglas sheet (corresponding to a distance slightly greater than the build-up of the used radiation) at 100 cm from the source of radiation.
3. Place a 0.5 cm-thick bolus on each sample before the irradiation occurs to guarantee the backscattered radiation component and the charged particles' equilibrium.
4. Irradiate the cells (doses in the range 2 – 10 Gy) with a flat and symmetric (± 2%) 20 x 20 cm₂ radiation field and a dose-rate of 3 Gy/min.
   NOTE: Control "sham"-irradiated cells underwent the same procedural conditions of the irradiated ones, without entering the irradiation room (received dose: 0 Gy).
   CAUTION: Ionizing radiation-producing devices must be used only by specialized personnel.

2. Trans-Epithelial Electrical Resistance (TEER)

1. Seed 5 × 10⁵ Caco-2 cells one week prior to irradiation in 6-well plate co-culture inserts (polyethylene terephthalate (PET), 2 x 10⁶ pores/cm2).
2. 1 h before irradiation, measure the TEER with a voltmeter/ohmmeter.
3. Irradiate cells as described in steps 1.1 – 1.4.
4. Incubate Caco-2 cells in the presence/absence of co-culture with PBMC at 37 °C in a humidified atmosphere containing 5% CO₂.
5. For the first few hours post-irradiation, measure TEER every hour, and subsequently every 3 hours up to 48 hours.