Iscove s modified dulbecco s medium

APCs were cultured in supplemented RPMI-1640 culture medium (Sigma). Sorted human T cells were cultured in Iscove’s Modified Dulbecco’s Medium (Sigma) containing recombinant IL-2 (1,000 U/mL) and 5% human serum. CD14⁺ monocytes were supplemented with human IL-4 (500 U/mL) and 50 ng/mL human GM-CSF (Peprotech) upon monocyte isolation and were fully differentiated on day 5. MoDCs pulsed with αGC were cocultured with the autologous CD14⁻ fraction. From this, iNKT cells were sorted and expanded as previously described. BM cells were plated at 2 million cells per well in a 6-well plate. Medium was replenished with fresh GM-CSF (20 ng/mL) every 2 d for 5 to 7 d for differentiation into CD11c⁺ BMDCs.

S. stercoralis L3i were concentrated by centrifugation, mixed in a 1:1 ratio with 2% low melt agarose solution (Gemini Bio, West Sacramento, CA, USA) and poured into a petri dish. Parasite-agar mixture was allowed to harden, and the L3i that migrated out of the agarose were collected and then concentrated by centrifugation in 10 ml of NI media consisting of a 1:1 mixture of NCTC-135 and Iscove’s Modified Dulbecco’s medium (Sigma Aldrich, St. Louis, MO, USA) with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 mg/ml levofloxacin (Levaquin; Akorn, Inc., Lake Forest, IL, USA) and 0.1 mg/ml gentamicin sulfate (EMD Millipore Corp., Billerica, MA, USA). L3i were then concentrated and washed 5 times in NI media. In the initial experiments humanized mice were infected with a low dose of 500 L3i injected subcutaneously every 7 days for 4-weeks to determine parasite survival in humanized mice. In the coinfection and steroid treatment experiments, humanized mice received a single infection of 5,000 L3i at 11–12 weeks post HSC engraftment. Steroid treated mice receive 2 mg of methylprednisolone acetate (MPA; Pfizer, New York, NY, USA) intraperitoneally once per week starting from time of the S. stercoralis infection until the end of the experiment [Fig 1].

The three cell lines used were all cultured in conformity with the sterile technique and the standard mammalian cell culture protocols under a 5% CO₂ atmosphere at 37 °C.
Lymphocyte cell line (IST-EBV-TW6B) was purchased from the cell bank IRCCS AOU San Martino IST (Italy). Cells were cultured in advanced RPMI 1640 culture medium (Gibco) with 20% of heat inactivated fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Sigma) and 1% of l-Glutamine 200 mM (Lonza) in 75 cm² not treated cell culture flasks (Corning) maintaining the cell density between 9 × 10^4–5 cells/mL.
Daudi cells (ATCC® CCL­213™), originating from a Burkitt’s lymphoma patient, were obtained from American Type Culture Collection (ATCC). Cells were grown in RPMI 1640 culture medium (ATCC) supplemented with 10% of heat inactivated FBS (ATCC), 1% P/S (Sigma) in 75 cm² not treated cell culture flasks (Corning) with a cell density between 3 × 10^5–6 cells/mL.
HL60 cells (ATCC® CCL-240™), from an acute myeloid leukemia patient, were purchased from ATCC. They were maintained in Iscove’s Modified Dulbecco’s Medium (Sigma) with 20% heat inactivated FBS (Sigma), 1% Glutamine (Sigma), 1% P/S (Sigma) in 75 cm² not treated cell culture flasks (Corning), adjusting cell density to 1 × 10^5–6 cells/mL.

Lactobacillus helveticus strains SIM12, SIS16, 1734 and Lh43 isolated from Italian hard cheeses were included in this study. The strains were reactivated overnight at 37°C in MRS (De Mann Rogosa and Sharpe) broth (Merck, Darmstadt, Germany, catalog number 1.10661.0500). Lact. rhamnosus ATCC 7469, used as auxotrophic strains in folate production analysis, was grown overnight at 37°C in MRS (Merck). Salmonella enterica serovar typhimurium strain SL1344 (FB62) was provided by G. Dougan (The Welcome Trust Sanger Institute, Hinxton, UK) and grown in LB (Luria-Bertani) broth (Sigma, Milan, Italy, catalog number L3022). L. helveticus strains were grown to an OD600 = 0.6 in MRS medium at 42°C. For the experiments with human peripheral blood mononuclear and dendritic cells cultures were centrifuged and pelleted cells were washed twice in IMDM (Iscove’s Modified Dulbecco’s Medium) (Sigma, St. Louis, USA, catalog number I3390) broth to eliminate residual MRS medium. Washed cells were then incubated at 37°C for 18 h in IMDM. Strain FB62 was grown at 37°C in LB medium (Sigma). The obtained L. helveticus cultures were used as such or filter sterilized (0.45 μm, Merck Life Science, Milan, Italy). Cell-free culture supernatants were called ‘postbiotics’.

Whole blood was collected in EDTA-treated tubes, and mononuclear cells were isolated by centrifugation on Ficoll-Hypaque (Sigma-Aldrich, Saint Louis, MO, USA) density gradient. For infection with Epstein-Barr virus (EBV), mononuclear cells were exposed to supernatants of the B95.8 cell line, according to standard procedures [31 (link)]. Cells were cultured in Iscove's Modified Dulbecco's Medium (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 20% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA) and 1% L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA). The presence of RYR1 SVs in the EBV-immortalized lymphoblastoid cells from patients was confirmed by DNA sequencing. Compared to cell lines carrying other RYR1 SVs, the cell culture carrying the insertion variant prematurely displayed increased average cycle times and increased cell size, which are characteristic of replicative senescence in cultured cells [32 (link)]. At this stage, the cells were discarded.

Whole blood was collected in EDTA-treated tubes and mononuclear cells were isolated by Ficoll-Hypapue density gradient centrifugation. For infection with Epstein-Barr virus (EBV), mononuclear cells were exposed to supernatants of the B95.8 cell line according to standard procedures20 . Cells were cultured in Iscove’s Modified Dulbecco’s Medium (13390, Sigma-Aldrich) supplemented with 20% fetal bovine serum (CH30160,03, Hyclone) and 1%L-glutamine (G7513, Sigma-Aldrich).