Fitc conjugated isotype control

U-CH2 and Chor-IN-1 cells were collected by trypsinization and recovered in their culture medium at 37 °C and 5% CO₂ for 30 minutes, then washed with PBS containing 1% FCS (staining buffer) and counted. 0.5 × 10⁶ cells of each cell line were resuspended in 100 µl staining buffer and stained with 10 µl PE-conjugated mouse anti-human CD24 or its correspondent PE-conjugated isotype control (BD Biosciences, San Jose, CA, USA) for 20 minutes at room temperature. Samples were washed with staining buffer, fixed with 1% formaldehyde for 10 minutes at 37 °C and permeabilized with 90% methanol for 30 minutes on ice. After washing with staining buffer, 10 µl of FITC-conjugated mouse anti-cytokeratin (CAM5.2) or its correspondent FITC-conjugated isotype control (BD Biosciences, San Jose, CA, USA) were added and incubated for 20 minutes at room temperature. Samples were washed with staining buffer, then acquired and analyzed with a FACSCalibur cytometer and CellQuest software (BD Biosciences, San Jose, CA, USA). Analysis was performed on 10,000 events, gating out debris and doublets.

MSCs isolated from cord blood were labeled with the following antibodies: FITC-conjugated human CD14 (cat# 555397), CD45 (cat# 555483), HLA-DR (cat# 555811), PE-conjugated human CD73 (cat# 550257), CD166 (cat# 559263, BD Biosciences), CD90 (cat# 12-0909-42), and CD105 (cat# 12-1057-42, Invitrogen). Isotype controls were also included: PE-conjugated Isotype Control (cat# 555743) and FITC-conjugated Isotype control (cat# 555573, BD Biosciences). Stained cells were analyzed by flow cytometry on a MACSQuant instrument (Miltenvi Biotec, Bergisch Gladbach, Germany).

NK degranulation assays were performed in a similar manner to that described previously [39] , [81] (link). Briefly, PBMC (approved by the Cardiff University School of Medicine Ethics Committee Ref. no: 10/20) and incubated overnight with IFN-α (1000 IU/ml) and IL-15 (15 ng/ml, Milenyi Biotech). PBMC (0.5–1×10⁶) were incubated for 6 h with 0.5–1×10⁵ fibroblast targets per well in a 96 well plate at an effector∶target (E∶T) ratio of 10∶1, with the addition of 3 µl per well FITC-conjugated anti-CD107 antibody (cat. no. 555800, clone H4A3, BD Biosciences) or 3 µl per well FITC-conjugated isotype control (cat. no. 555748, BD Biosciences), adding 1 µl/well BD GolgiStop (BD Biosciences) 1 h after beginning the incubation. (For antibody blocking experiments, targets were pre-incubated with anti-MICA (Clone 159277, mouse IgG2B, MAB1300, R&D Systems) or isotype control (MICB non-blocking antibody, Clone 236511, mouse IgG2B, MAB1599, R&D Systems) antibodies at a concentration of 10 µg/ml for 30 min prior to incubation with PBMC). PBMC were harvested and stained with conjugated antibodies against CD3 (anti-CD3 PE-Cy7, cat. no. 737657, Beckman Coulter, High Wycombe, UK) and CD56 (anti-CD56 PE, cat. no. A07788, Beckman Coulter), and fixed in 2% PFA before analysis by flow cytometry (BD Biosciences Accuri C Flow) (Fig. S10A).