A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The following protocol involves human blood withdrawal from healthy volunteers. Donors provided written informed consent prior to enrollment. This procedure is in accordance with the Helsinki Declaration and blood withdrawals were performed by a professional healthcare assistant.

1. Cell Culture and Co-culture Set-up

1. One week before the irradiation, prepare a Caco-2 cell suspension containing 2.5 × 10⁵ cells/mL in fresh RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
2. Seed 2 mL of cell suspension in sterile 1 µm-pore diameter cell culture inserts for 6-well plates and put the insert into a 6-well plate.
   NOTE: Cell culture inserts might need to be activated by incubation with sterile complete medium prior to cell seeding. In this case, culture media should be discarded and replaced with cell suspension media.
3. Add 3 mL of fresh RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin in each bottom compartment, and culture the cells at 37 °C in an incubator with humidified atmosphere containing 5% CO₂.
4. On the same day of Caco-2 cell irradiation, collect human whole blood in commercially available lithium-heparin coated 6 mL tubes (tube size: 13 x 100 mm).
5. Subsequently, isolate peripheral blood mononuclear cells (PBMC) by using Ficoll gradient. To separate PBMC, put 25 mL of Ficoll in a 50 mL conical centrifuge tube and layer an equal volume of whole blood diluted 1:1 with RPMI1640 onto the Ficoll surface.
   NOTE: A normal healthy donor usually has approximately 4 – 10 × 10⁶ PBMC/mL.
6. Centrifuge the 50 mL tubes at 400 x g for 30 min at room temperature.
7. Gently collect the PBMC at the interface between Ficoll and plasma by aspiration with a Pasteur pipette and put them in a 15 mL conical tube.
8. Wash the PBMC twice by adding 10 mL of phosphate buffered saline (PBS) and centrifuging PBMC at 250 x g for 10 min.
9. Culture PBMC for a maximum of 3 – 5 h in T25 cm² flasks in complete RPMI1640 media, as described before, at 37 °C in a humidified atmosphere containing 5% CO₂.
   NOTE: Collect PBMC on the day of the experiment and seed 2 × 10⁶ cells/well, suspended in 3 mL of complete RPMI1640 medium, in the bottom compartment of the co-culture. Inserts with Caco-2 cells are transferred in PBMC-containing wells 30 min after their irradiation.PBMC collected from whole blood cannot be maintained in culture for more than approximately 72 h, if not stimulated with e.g. phytohaemagglutinin (PHA), after which cells' viability is lost.
   CAUTION: The quality and safety of all blood and blood products must be guaranteed throughout the whole process.

2. 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-rays 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.

3. Cell Viability Assay (MTT Assay)

Note: Caco-2 cell metabolic activity was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay¹⁴.

1. Seed 2 × 10⁵ Caco-2 in a 24-well plate 24 h before irradiation in 1.25 mL of complete medium.
2. Irradiate the cells as described in steps 2.1 – 2.4 then incubate the cells at 37 °C in a humidified atmosphere containing 5% CO₂.
3. 21 h after irradiation, add 100 µL of 5 mg/mL MTT solution and keep the cells at 37 °C in a humidified atmosphere containing 5% CO₂ for 3 h.
4. Discard the supernatant and wash the cells with 1 mL of PBS.
5. Dissolve formazan crystals by adding 500 µL of dimethyl sulfoxide (DMSO) in each well and evaluate the absorbance with a multi-well plate reader at λ = 570 nm.
6. Repeat step 3.3 – 3.4 – 3.5 – 3.6 at 45 h after irradiation.
   NOTE: Results are shown as a perturbation from the corresponding sham condition, which is normalized to 100%.
   CAUTION: DMSO is inflammable and irritating agent for skin, eyes and respiratory system (GHS07, GHS08). In case of contact with eyes, wash immediately with abundant water and seek medical advice. MTT is an irritating agent for skin, eyes and respiratory system (GHS07, GHS08). In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.

4. Percentage of Viable Cells Determination (Trypan Blue Dye Exclusion Assay)

NOTE: Percentage of viable cells was assessed by Trypan Blue Dye Exclusion assay.

1. Seed 2 × 10⁵ Caco-2 in 24-well plate 24 h before irradiation in 1.25 mL of complete medium.
2. Irradiate the cells with doses in the range 0 – 10 Gy (see steps 2.1 – 2.4) then incubate the cells at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 – 48 h.
3. After the two chosen time points, wash the cells with PBS, discard it, then add 100 µL of Trypsin-ethylenediaminetetraacetic acid (EDTA) solution. Put the cells at 37 °C in a humidified atmosphere containing 5% CO₂ for 2 min then add complete medium to arrest the enzymatic reaction.
4. Collect the cells in a 1.5 mL-tube and centrifuge the tube at 500 x g for 5 min. Discard the supernatant and re-suspend the cells in 50 µL of PBS.
5. Mix the cell suspension with 50 µL of Trypan Blue dye solution and incubate the mixture for 3 min at room temperature.
6. Count the stained (non-viable) and unstained (viable) cells with a Bürker chamber.

5. 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 2.1 – 2.4.
4. Incubate Caco-2 cells in 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 h up to 48 h.

6. Western Blot Analysis of Claudin-1, Occludin, Afadin, ZO-1, ZO-2, NF-kB, and XIAP

1. Seed 5 × 10⁵ cells 1 week before X-rays exposure in 6-well plate co-culture inserts (PET, 2 x 10⁶ pores/cm²) and irradiate cells as described in steps 2.1 – 2.4.
2. Incubate Caco-2 cells in presence/absence of co-culture with PBMC at 37 °C in a humidified atmosphere containing 5% CO₂.
3. 48 h after irradiation, prepare Caco-2 and PBMC cell lysates by treating cells with cell lysis buffer and store samples at -20 °C.
   NOTE: the protocol can be paused here.
4. Quantify total protein amount by bicinchoninic acid (BCA) method.
   NOTE: the protocol can be paused here.
5. Mix equal amounts of total proteins with Laemli Sample Buffer added with β-mercaptoethanol (final concentration of β-mercaptoethanol is 5%), heat the samples at 95 °C for 5 min and spin them at 10,000 x g.
6. Load samples in a 4 – 20% precast gel and perform the electrophoresis at 120 V for 1 h. Transfer proteins on a polyvinildiene fluoride (PVDF) membrane.
7. Block non-specific binding sites by incubating PVDF membrane at room temperature with 5% non-fat dry milk (NFDM) in PBS added with 0.2% Tween-20 (PBT). Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.
8. Incubate the membrane with gentle agitation for 1 h at room temperature prior to being placed overnight at 4 °C with the following primary antibodies: anti-claudin-1, anti-ZO-1, anti-ZO-2, anti-afadin, anti-occludin, anti-NF-kB, anti-XIAP, and anti-actin. Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.
9. Incubate the membranes with Horseradish Peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature with gentle agitation. Wash the membrane three times with 10 mL of 0.2% PBT for 5 min.
10. At room temperature, develop photographic films by incubating the membranes with enhanced chemo-luminescent kit.
11. Acquire photographic films with a scanning system and quantify the obtained bands with suitable image analysis software.
    NOTE: The evaluation of Claudin-1, Occludin, Afadin, ZO-1, and ZO-2 is performed in Caco-2 cells. The evaluation of NF-kB and XIAP is performed in PBMC.
    CAUTION: β-mercaptoethanol is toxic (GHS05, GHS06, GHS08, GHS09). Do not breathe vapors, avoid release into the environment, and wear individual protection devices and respiratory protection. If swallowed, immediately ask for medical advice.

7. Statistical analysis

1. To determine whether the radiation exposure and the co-culture induce a statistically significant perturbation, perform a two-way analysis of variance (ANOVA) test with multiple comparisons for repeated measurements (with Bonferroni post-hoc tests to compare replicate means).
2. If not otherwise stated, calculate the statistical significance (p) by two-tailed Student's t-test. Each value is the mean of ≥3 independent experiments ± Standard Error of the Mean (SEM).