Facscanto system

HeLa cells (ATCC reference CCL-2) were cultured into a treated 24 well plate (6 × 10⁴ cells per well) with Minimum Essential Medium (MEM, Gibco, Thermo Scientific) supplemented with 10 % FBS and 2 mM Glutamax (MEMα-FBS-G) 24 h at 37 °C and 5 % CO_2. After 24 h, medium was removed and cells were immediately washed twice with DPBS. Afterwards, 5 µg of VP1GFP IBs along with Optipro medium (Gibco, Thermo Scientific) supplemented with 2 mM l-Glutamine were added. The plate was incubated 24 h at 37 °C and after incubation, 250 µL of trypsin (1 mg/mL) were added for 15 min to detach cells and to remove IB protein that might be externally associated. Trypsin was inactivated by the addition of 750 µL of MEMα-FBS-G and the cells were centrifuged at 1200×g, at 4 °C for 5 min. Obtained samples were analyzed on a FACS Canto system (Becton–Dickinson, Franklin Lakes, NJ, USA) using a 15 W argon-ion laser at 488 nm excitation and fluorescence emission was measured with a 530/30 nm band pass filter.

H460 cells were infected with Ad-LacZ and Ad-LETM1 for 24 h. Then cells were collected and stained with JC-1 dye (Molecular probes, Invitrogen, Eugene, USA) for 30 min. The cells were treated with 30 μM CCCP for 10 min as a positive control to induce mitochondrial depolarization before staining with the dye. Finally, the cells were analyzed by FACS Canto system (Becton Dickinson, Oxford, UK).

MDAMB361 and BT474 cells were exposed to docetaxel (100 nM) for 6 to 72 hours or left untreated, collected and incubated at 4°C with anti-MICA, -MICB, -ULBP1, -ULBP2 (R&D Systems), followed by incubation with anti-mouse Alexa Fluor 448-conjugated reagent (Invitrogen, Waltham, MA, USA). Samples were analyzed by gating on live cells using the FACSCanto system (Becton-Dickinson, San Jose, CA, USA) and BD FACSDiva™ software (BD Bioscience).
PBMCs isolated from patients or healthy donors were analyzed using monoclonal antibodies to human antigens, including PE-anti-CD16 (3G8, BD Bioscience), PECy5-anti-CD56 (B159, BD Bioscience), APC-eFluor780-anti-CD3 (SK7, eBioscence, San Diego, CA, USA), FITC-anti-CD69 (FN50, BD Bioscience) and APC-NKG2D (ON72, Beckman Coulter, Miami, FL, USA).
Total splenocytes or purified Gr1⁺ myeloid cells from mouse spleens were analyzed using mouse monoclonal antibodies, including PE-anti-CD49b (DX5, eBioscience), APC-anti-NKG2D (CX5, eBioscience), PE-anti-CD11 (MI/70, BD Bioscience), FITC-anti-Ly6G (1A8, Mylteni Biotec) and APC-anti-Ly6C (HK1.4, eBioscience). Samples were analyzed by gating on physical parameters using the FACSCanto II system (Becton-Dickinson) and FlowJo software (Tree Star Inc, San Carlos, CA, USA).

After co-culture for 3 days, cell suspensions were incubated with FITC-conjugated anti-human CD4 and PE-conjugated anti-human CD25 (Biolegend, San Diego, CA, USA) for 30 min at 4 °C and washed twice with 2 ml of phosphate-buffered saline (PBS) pH 7.4 containing 1 % bovine serum albumin (BSA). Intracellular staining for Foxp3 was then performed with APC-conjugated anti-human Foxp3 (eBioscience, San Diego, CA, USA) for 60 min and then washed with PBS/BSA. The supernatants were discarded and cells were resuspended in 0.2 ml PBS/BSA. Data were acquired with a FACSCanto system (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using Flowjo software (Tree Star, Inc., Ashland, OR, USA). The expression levels of CD4, CD25 and Foxp3 were evaluated by calculating the percentage of cells expressing each protein.