Tryple enzyme

Mouse KCs were isolated from the skin of newborn mice, as described [28 (link)], except that the epidermis was dissociated following 0.05% DNase addition (Sigma-Aldrich, Italy). 5*10^5 KCs were plated in T25 cell culture flask coated with mouse type IV collagen (BD Biosciences, Belgium) and grown in Cnt-57 progenitor cell-targeted medium (CELLnTEC, Switzerland). At subconfluence, cells were detached using TrypLE enzyme (Thermo Fisher Scientific, MA USA) and either used for the experiments, or seeded for further passages at 2*10^5 cells in 5 mL medium.

Matrigel (Corning Life Sciences, USA) covered plates were used for culturing hESC in monolayers in conditioned human embryonic stem cell medium (CHESM, see below). After reaching the monolayer, the cells were detached by the TrypLE enzyme (Thermo Fisher Scientific, USA), collected, washed and counted in Neubauer hemocytometer chamber. 300,000 cells were spinned down in 1.5 mL tubes, the medium was aspirated, the cell pellet resuspended in remaining 3–5 μL medium and used in experiments. To prepare the CHESM, we derived mouse embryonic fibroblasts (MEFs) from 12.5-day murine embryos using the standard protocols available in our lab. The MEFs were then frozen and stored at liquid nitrogen for subsequent medium preparations. The fresh HES medium was incubated in the plates with MEF monolayers for 24 h to obtain CHESM.

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol (10-02214) was approved by the Ethics Committee of the UCSF Medical Center (CA, USA). Several stem cell lines, including hiPSC UEFhfiPS1.4 [32 (link)], hESC CCTL12 [13 (link),33 ,34 (link)] and hiPSC UB47 [12 (link),33 ], were maintained as colonies on mitotically inactivated mouse embryo fibroblast (MEF) feeder in HES medium with 10 ng/mL human FGF2 or single cells on Matrigel hES-qualified (Corning, Corning, NY, USA, ref: 354277) as previously described [12 (link)]. Briefly, the colonies were manually dissected using a needle and passaged every 4–6 days whereas the single cells were enzymatically dissociated using TrypLE enzyme (Gibco, Waltham, MA, USA, ref: 12604-013) and passaged every 4 days.

All chemicals were of analytical grade. The following chemicals were from Merck Sigma-Aldrich (Milan, Italy): QA (Cat # P63204), L-glutamate (G1626), glycine (G7403), and probenecid (Cat # P8761). Ketamine was from Merial (Cat # Imalgene 1000). Esmethadone hydrochloride (CAS: 15284-15-8) was from SpecGx (Webster Groves, MO, USA; Cat # 8514). Fluo-4 (F14202) was from Invitrogen (Milan, Italy). The following cell culture chemicals were from Gibco Life Technologies (Milan, Italy): Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; Cat # 21331), heat-inactivated fetal bovine serum (Cat # 10500), penicillin-streptomycin (Cat # 15140), L-glutamine (Cat # 25030), MEM nonessential amino acids (Cat # 11140), blasticidin (Cat # A11139), G418 (Cat # 10131), Zeocin™ (Cat # R250), and TrypLE™ enzyme (Cat # 12604). Hygromycin B (Cat # 10687) was from Invitrogen. Gentamicin sulphate concentrations were expressed in μg/mL as gentamicin sulphate was a mixture of three major components designated as C1, C1a, and C2. Calculated and corrected for purity, the molecular weight was 681.58 Da, such that 100 µg/mL corresponded to 146.7 µM gentamicin sulphate.

PBMCs (1 × 10⁵ cells per well) were either left non-stimulated or were stimulated with anti-CD3/CD28 mAb-conjugated beads (11131D; Gibco; 1 × 10⁵ beads per well) for 24 h, together with anti-IFN-γ neutralizing mAb (NIB42, 10 μg/ml; eBioscience) or isotype control (MOPC-21, 10 μg/ml) in a 96-well U-bottom plate. The non-adherent cells were transferred to a 96-well V-bottom plate by pipetting. The residual adherent cells were detached by incubation with the TryPLE enzyme (Gibco) at 37°C for 5 min and were then transferred to the same 96-well V-bottom plate. Cells were washed once with FACS buffer, surface-stained by incubation at 4°C for 1 h with various antibodies (anti-CD3-FITC [UCHT1, 1:100; Cytek], anti-CD19-BV650 [HIB19, 1:100; BD], anti-CD56-V450 [B159, 1:50; BD], anti-CD14-Spark NIR 685 [63D3, 1:100; BioLegend], anti-CD16-PE/Dazzle 594 [3G8, 1:100; BioLegend], anti-HLA-DR-APC/Fire 810 [L243, 1:100; BioLegend], anti-CD123-BV480 [9F5, 1:100; BD], anti-CD11c-Alexa Fluor 700 [Bu15, 1:1,000; BioLegend], anti-PD-1-BB700 [EH12.1, 1:100, BD], and anti-PD-L1-BV711 [29E.2A3, 1:100; BioLegend] or an isotype control [MPC-11; BioLegend]), washed, stained with 7-AAD (1:200), and acquired with an Aurora cytometer (Cytek). Data were analyzed with FlowJo software. The median fluorescence intensity (MFI) of PD-L1 was used as a readout.

All chemicals were of analytical grade. Dextromethorphan and (±)-ketamine were from Merck Sigma-Aldrich (Milan, Italy). Memantine and (+)-MK-801 were from Bio-Techne/Tocris (Milan, Italy). REL-1017 (dextromethadone HCl) was from SpecGx (Webster Groves, MO, USA).
Cell culture consumables were from Gibco Life Technologies (Milan, Italy), including Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12; Cat # 21331), heat-inactivated fetal bovine serum (FBS; Cat # 10500), penicillin-streptomycin (Cat # 15140), L-glutamine (Cat # 25030), MEM nonessential amino acids (NEAAs; Cat # 11140), blasticidin (Cat # A11139), G418 (Cat # 10131), Zeocin™ (Cat # R250), and TrypLE™ enzyme (Cat # 12604). Hygromycin B (Cat# 10687) and Fluo-4 (F14202) were from Invitrogen (Waltham, MA, USA).

The hiPSC lines were generated from a blood sample of a 32-year-old female patient exhibiting the short-coupled PMVT at rest and carrying the RyR2-H29D mutation, as previously characterized [6 (link)] with the consent of the index patient and following the approval by the Institutional Review Board of Weill Cornell Medicine (NY, USA). As previously described, the RyR2-H29D mutation was corrected using CRISP/Cas9 technology thus generating an isogenic control hiPSC line (PMVT-29-corrected) [7 (link)]. The hiPSC lines were maintained on hES-qualified Matrigel (Corning, 354,277) at standard conditions (21% O₂, 5% CO₂ and 37 °C) and were enzymatically dissociated using TrypLE enzyme (Gibco, ref: 12,604–013) and passaged every 4 days. As 2 independent clones of PMVT RyR2-H29D hiPSC (C1, C3) tested in a previous study [7 (link)] exhibited identical properties, here we focused on the C1 clone only.