Mem amino acids

African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) supplemented with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies) at 37°C with 5% CO₂. Baby hamster kidney cells constitutively expressing the T7 polymerase (BSR T7/5) [56 (link)] were grown in GMEM media (Gibco-Life Technologies) supplemented with 10% FBS and 2% MEM amino acids (Gibco-Life Technologies). Every second cell passage, 2% of gentamicin (Quality Biological) was added to maintain selection for the T7-polymerase-expressing cells.

PBMCs were isolated by Ficoll-Paque density gradient centrifugation. CD14⁺ monocytes were obtained from PBMCs by positive selection using microbeads according to the manufacturer's protocol(Miltenyi Biotec). The purity of monocytes from healthy donors was greater than 90%, and the purity from CLL patients was greater than 85%. Although monocytes can be further subdivided based on relative expression of CD14 and CD16, given that approximately 90% display the classical phenotype (CD14⁺CD16⁻), and that monocytes represent only approximately 1% of PBMC in CLL patients (limiting the ability to analyze subtypes), we chose to use all CD14⁺ cells for these experiments. To generate DCs, CD14⁺ monocytes were cultured in RPMI 1640 complete medium (10% heat inactivated fetal bovine serum, 1% GlutaMAX, 1mM sodium pyruvate, 0.5% MEM-amino acids, 1% MEM-Vitamin, 0.07 mM β-ME, 1% penicillin/streptomycin; Gibco^®, Grand Island, NY, USA) supplemented with GM-CSF (50 ng/ml; PeproTech, Rocky Hill, NJ, USA) and IL-4 (50 ng/ml; PeproTech). After 5 days, immature Mo-DCs (Mo-iDCs) were induced to mature with LPS (Escherichia coli; 100ng/mL; Sigma-Aldrich, St. Louis, MO, USA). At day 6, mature Mo-DCs (Mo-mDCs) were harvested for further experiments.

HeLa (kindly provided by Pietro De Camilli, Yale University) and RAW 264.7 (ATCC) cells were maintained in DMEM + 10 % FBS + 1 % penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C with 5 % CO2. Details of cell lines are summarized in Supplemental Table 1. Experiments to test the effect of amino acids on cells were performed as per our previous studies (Amick et al., 2016 (link); Amick et al., 2020 ; Meng and Ferguson, 2018 (link); Petit et al., 2013 (link)). In brief, cells were washed 2 X in PBS and incubated in RPMI without amino acids (US Biological) for 1 h and then were shifted to RPMI with MEM Amino Acids (Gibco) at the indicated times. BX-795 (Cayman, dissolved in DMSO) and LLOMe (Cayman, dissolved in DMSO) were added to growing cells at the indicated concentrations and compared with cells treated with an equivalent amount of DMSO alone. Details of growth media and drugs are summarized in Supp. Table 2.

Acinar cells were isolated as previously described [2 (link)]. Briefly, mice or rats (Charles River Laboratories, Wilmington, MA) were euthanized by CO₂ inhalation. The pancreas was minced in buffer-A [10 mM HEPES (pH 7.4), 95 mM NaCl, 4.7 mM KCl, 0.6 mM MgCl₂, 1mM NaH₂PO₄, 10mM glucose, 2mM glutamine, 0.1% bovine serum albumin, and 1× MEM amino acids (GIBCO-BRL, San Jose, CA)] and washed three times. Cells were then digested for 1h at 37°C in buffer-A containing 50 U/ml of type IV collagenase (Worthington, Freehold, NJ) with sustained shaking. The digest was filtered through a 200 μm mesh (Sefar American, Depew, NY), and the resulting groups of acinar cells were distributed in a 24-well Falcon tissue culture plate (Becton Dickinson, Franklin Lakes, NJ) and placed in a water bath shaking at 90-rpm under constant oxygen flow to recover. Human pancreatic acinar cells were prepared as previously described[17 (link)].

Imaging of NO and CBF was as described [28 (link)]. For NO, ALI cultures were loaded with DAF-FM by incubation in 10 μM DAF-FM diacetate (ThermoFisher) on the apical side in HBSS plus 5 μM carboxy-PTIO (to scavenge baseline NO; Cayman Chemical) for 90 min. For submerged CFBEs on 8-well chambered coverglass (CellVis, Sunnyvale, CA, USA), loading was the same but for 45 min instead of 90 min. After washing, imaging was performed using an IX-83 microscope (10x 0.4 NA PlanApo objective, Olympus Life Sciences, Tokyo, Japan) with LED illumination (Excelitas Technologies LED120Boost), 16-bit Orca Flash 4.0 sCMOS camera (Hamamatsu, Japan), standard FITC filter set (470/40 nm excitation, 495 lp dichroic, and 525/40 nm emission; 49002-ET, Chroma Technologies) and MetaFluor (Molecular Devices, Sunnyvale, CA, USA). For cilia beating, cultures were maintained at ~28 °C in DPBS (+1.8 mM calcium) on the apical side and HEPES-buffered HBSS supplemented with 1× MEM amino acids (Gibco) on the basolateral side. Sisson-Ammons Video Analysis software was used to measure whole-field CBF. Diphenhydramine and all other reagents used were from Millipore Sigma unless otherwise specified.

JM-H for CSSC culture: DMEM (1 g/L D-glucose, Gibco 10567-014), MCDB 201 (Sigma-Aldrich M6770), added with insulin-transferrin-selenite (ITS, 0.5×, Gibco 41400-045), AlbuMAX I (1 mg/mL, Gibco 11020-021), L-ascorbate-2-phosphate (0.5 mM, Sigma-Aldrich A8960), recombinant human EGF (10 ng/mL, Sigma-Aldrich E9644), recombinant human PDGF-BB (10 ng/mL, R&D 520-BB), dexamethasone (10 nM, Sigma-Aldrich D4902), penicillin/streptomycin (BioWhittaker, Lonza, Walkersville, MD, USA), and 2% pooled human serum (Innovative Res.).
ERI for CSK culture: DMEM/F12 (Gibco 10565-018), MEM amino acids (Gibco 11130-051), MEM non-essential amino acids (Gibco 11140-050), ITS (0.5×), AlbuMAX I (1 mg/mL), L-ascorbate-2-phosphate (0.5 mM), recombinant insulin growth factor 1 (10 ng/mL, Sigma-Aldrich I3769), Y27632 (10 nM, Chemdea CD0141), human amnion stromal extract (5 μg protein/mL, preparation following the method of Yusoff et al. [24 (link)], and penicillin/streptomycin.