Nk isolation kit

NK cells and CD4 T cells were isolated from PBMC (>95% purity and viability) (NK isolation kit and CD4 T cell isolation kit respectively; Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions.

iPSC-ECs were treated or not for 48 h with IFNγ. Then, 48 h later, cells were detached and 40,000 cells were seeded in a U bottom 96-well plate together with NK cells, isolated using the NK isolation kit (Miltenyi Biotec, S.r.l., Bologna, Italy) in a 1/1 ratio in RPMI supplemented with 10% human AB serum. After 24 h of coculture, cell lysis was assessed using the CytoSelect LDH Cytotoxicity Assay kit (Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Results were expressed as percentages of specific NK-induced cell lysis, considering the LDH release obtained via cell incubation with Triton X-100 solution as the maximal cell lysis.

Peripheral blood mononuclear cells were isolated from each patient’s blood sample by Ficoll lymphoprep density gradient centrifugation (BD science, Tianjin, China). NK cells were purified with a NK isolation kit (MiltenyiBiotec, Germany) to a ≥98% CD56 expression and <1% CD3 expression. Cell viability exceeded 90%, as determined by trypan blue staining. The separated NK cells were cultured in 6-well plates at a density of 5 × 10⁵ cells/ml in RPMI 1640 medium (GE Health Life Science, United States), supplemented with 15% of fetal bovine serum (Gibco Invitrogen), 100 U/ml of penicillin-streptomycin (Gibco Invitrogen), and 500 U/ml of interleukin-2 (peprotech, United States). K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Zhejiang Tianhang Technology, Zhejiang, China) and 100 U/ml of penicillin-streptomycin (Gibco Invitrogen). Because NK cells were not cultured under continuous proliferation conditions and a longer culture time (>48 h) would induce widespread apoptosis of NK cells, we used a 24 h NK cells culture period.

NK92-MI cells (ATCC CRL-2408), were grown and cultivated in minimum essential medium (MEM) alpha medium (Life Technologies, Carlsbad, CA) supplemented with 12.5% FBS and donor horse serum (Atlanta Biologicals, Lawrenceville, GA), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, and 0.02 mM folic acid. All cells were cultured at 37°C in a humidified 5% CO₂/95% air environment. Peripheral blood mononuclear cells (PBMCs) were isolated from ethylene-diamine-tetra-acetic acid (EDTA)-treated whole-blood samples by Histopaque-1077 (Sigma Chemicals, St. Louis, MO) density gradient centrifugation using LeucoSep tubes (Greiner, Monroe, NC) from healthy individuals with prior approval from the Office of Protection of Human Subjects, The North Texas Regional Institutional Review Board (IRB# 20–28) of UNT Health Science Center, Fort Worth, TX. A written consent was obtained from healthy donors and no minors were involved in this study. Primary NK cells were isolated from the PBMCs using NK isolation kit (Miltenyi Biotec, San Diego, CA) and the purity was determined by flow cytometry using anti-human CD56 mAb.

Human peripheral blood samples were obtained with informed consent from a panel of seven healthy donors (male:female, 4:3) aged between 24 and 41 from the department. The study had been approved by the School Human Studies Ethics Committee. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque gradient centrifugation (Lymphoprep, PAA laboratories, Pasching, Austria). Total NK cells were isolated from PBMCs using NK isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. After the isolation, the purity of the CD56⁺CD3⁻ NK cell preparation was routinely more than 90%. Purified human NK cells were cultured in completed RPMI-1640 (10% heat-inactivated FCS, 50 µM 2-mecaptoethanol, 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin) supplemented with 50 pg/ml recombinant human IL-2 at 37°C in 5% CO₂. In some experiments, iC3b-containing serum (10%) or iC3b-bound apoptotic cells (at 1:1 NK-apoptotic cell ratio) were added into culture medium for indicated time period.

Healthy donors’ peripheral blood buffy coats were obtained from San Matteo Hospital, Pavia, Italy, whereas intestinal specimens were obtained from IRCCS Policlinico Ospedale Maggiore, Milan, Italy. Institutional Review Board (Milan, Area B) approved the study with permission number 566_2015. All methodologies were under full compliance with the Declaration of Helsinki, and informed consent was obtained from all subjects.
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll‐Paque as for standard protocol. Natural killer cells were isolated from PBMCs using the NK Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human lamina propria mononuclear cells (LPMCs) were isolated as previously described [53 (link)]. Briefly, the dissected intestinal mucosa was depleted of mucus and epithelial cells in sequential washes with Hanks’ Balanced Salt Solution (HBSS, Euroclone, Pero, Italy) containing DTT (0.1 mmol·L⁻¹, Sigma‐Aldrich, St Louis, MO, USA) and EDTA (1 mmol·L⁻¹, Sigma‐Aldrich) and then digested with collagenase D (400 U·mL⁻¹, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 5 h at 37 °C. LPMCs were then separated with a Percoll gradient and cultured in RPMI‐1640 medium containing 5% human serum (Sigma‐Aldrich), penicillin/streptomycin, gentamicin, and 100 U·mL⁻¹ IL‐2 (Proleukin).