Countess

Cell concentration and cell integrity/viability were determined by trypan blue dye exclusion staining and the Countess^TM cell counter (Invitrogen, Thermo Fisher Scientific, Waltham, MA) for the species mixing experiment and MDMs. For rat liver immune cells both parameters were estimated by the NucleoCounter NC-200^TM device (Chemometec, Allerod, Denmark; acridine orange and DAPI staining).

Human gastric cell lines were cultured on 12-well plates at a density of 50,000 cells per well for 24 h and then treated with linsitinib, IGF-1R inhibitor (Selleck Chemicals, Houston, TX, USA), or equivalent volumes of vehicles to concentrations for exposure times indicated. The number of viable cells was counted by Trypan blue dye exclusion using the CountessTM automated cell counter (Invitrogen). The results were shown as a relative ratio to controls.

Dendritic cells were cultivated in a 24-well cell culture plate at a concentration of 2 × 10⁵ cells/mL with EVs at different concentrations, EV1 (8 × 10⁷ EVs/well), EV2 (1.7 × 10⁸ EVs/well) and EV3 (3.5 × 10⁸ EVs/well), during 4 and 24 h at 37°C with 5% of CO₂. Thereafter, the adherent cells were removed with a cell scraper and stained with 0.5% trypan blue stain (Invitrogen^TM), followed by counting using an automated cell counter Countess^TM (Invitrogen^TM), which measured trypan-negative, trypan-positive and total cells.

Spleen and MLN cells were isolated as previously described [39 (link)]. IEL suspensions from the proximal ¾ portion of the intestine were obtained following procedures adapted to neonatal rats and established previously in our laboratory [32 (link)]. They were later purified and cell number viability was determined using an automated cell counter after staining dead cells with trypan blue (Countess^TM, Invitrogen, Madrid, Spain).

The MLNs were placed in complete culture medium, containing Roswell Park Memorial Institute (RPMI 1640, Sigma-Aldrich, St. Louis, MO, USA) 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 1% l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin streptomicin (PenStrep; Sigma-Aldrich, St. Louis, MO, USA), and 0.05 mM 2-β-mercaptoethanol (Merck, Darmstadt, Germany). MLNs lymphocytes were obtained in sterile conditions by passing the tissue through a cell strainer (40 µm, BD Biosciences, San Diego, CA, USA). The cell suspensions were centrifuged (538 g, 10 min, 4 °C), and the pellet was resuspended with complete RPMI medium. The cell counts and viabilities were determined using an automated cell counter, after staining the cells with trypan blue (Countess^TM, Invitrogen, Madrid, Spain), following usual laboratory procedures, as described in Reference [22 (link)]. The lymphocytes were immediately used to characterize their phenotype, and to determine their ability to proliferate and secrete cytokines.

Jurkat lymphocytes and CY15 dendritic cells (2 × 10⁵ cell/ml) were cultured in 100 µl of complete RPMI culture media 24 h after FACS sorting in 96-well plates. A Countess^TM automated cell counter (Invitrogen) was used for cell size and viability by means of trypan blue exclusion. The AlamarBlue dye (Life Technologies) was used to evaluate proliferation following the manufacturer's instructions. In some experiments, increasing concentrations of puromycin were used to monitor the proliferation of LvKCNE4 and LvScramble CY15 cells. Values of control cells, cultured in the presence of FBS during 24 h after seeding, were considered to represent 100% proliferation.

Cells were cultured as tumor spheres in DMEM containing 20 ng/mL hEGF (R&D Systems, Minneapolis, MN), 10 ng/mL hbFGF (R&D Systems), 4 mg/mL heparin sulfate (Sigma, St. Louis, MO), 0.15% bovine serum albumin (BSA) (Sigma), and 1% penicillin G-streptomycin [19 (link)]. Sphere forming capacity (SFC) was determined by the ability to form three-dimensional spheroids in culture over a period of three to seven days. For assessment of long term proliferation and fold expansion, spheres were disassociated and passaged every 7 days using 0.05% trypsin-EDTA (Invitrogen). Sphere cultures were grown in low-adherent flasks (Nunc, Penfield, NY). Cells were counted using a CountessTM (Invitrogen) automated cell counter. To induce tumor stem cell differentiation, the cultured tumorspheres of HepG2 and Huh7 cells were in medium with 10% (v/v) FBS for seven days at 37 °C in a humidified chamber supplemented with 5% CO₂.