Rpmi 1640 with l glutamine

PBMCs from healthy young and older donors were plated at a volume of 1.0 × 10⁶ cells per well in a round 96-well plate in a volume of 100 ul of complete RPMI medium (RPMI 1640 with L-glutamine [Corning Cellgro, Manassas, VA] supplemented with 10% FBS and 13 [50 U] penicillin-streptomycin [Invitrogen, Carlsbad, CA]). For experiments involving LPS/ IFNg stimulation, PBMCs were stimulated for 6 or 24 h with 0.5 ug/mL LPS (Invivogen, Cat# TLRL-ebLPS) plus 40 ng/mL IFNg (Invivogen, Cat#300-134 P). For experiments involving empty LNP, PBMCs were stimulated for 6 or 24 h with 0.78 ug/mL eLNP. Following stimulation, PBMCs were analyzed via flow cytometry and supernatants were collected after stimulation and frozen at −80 °C

PBMCs from healthy young and older donors were plated at a volume of 1.0 × 10⁶ cells per well in a round 96-well plate in a volume of 100 µl of complete RPMI medium (RPMI 1640 with l-glutamine [Corning Cellgro, Manassas, VA] supplemented with 10% FBS and 13 [50 U] penicillin–streptomycin [Invitrogen, Carlsbad, CA]). For experiments involving LPS/IFN-α or IFN-γ stimulation, PBMCs were stimulated for 15 min, 45 min, or 24 h with 1 µg/ml LPS with 80 ng/ml (InvivoGen, Cat# TLRL-eblps) and IFN-γ (InvivoGen, Cat# 300-134P). For experiments involving RIG-I agonist, PBMCs were stimulated for 24 h with 500 ng/ml of a RIG-I ligand, 3p-hpRNA/LyoVec (InvivoGen, Cat# tlr-hprnalv). A LyoVec-only control was used for this stimulation. For experiments involving a cyclic GMP-AMP synthase (cGAS)-STING agonist, PBMCs were stimulated for 24 h with 75 µM of G10 STING agonist provided to us by Dr. Vincent DeFillipis at the Vaccine and Gene Therapy Institute at Oregon Health and Science University and DMSO control was used. Optimal concentrations of different TLR agonists were selected based on the median production of IL-6 and IFN-α and an 85% or more survival rate of monocytes. All PRR ligands were purchased commercially (InvivoGen, San Diego, CA) except for the G10 STING agonist. Where indicated, supernatants were collected after stimulation and frozen at − 80 °C.

The human bronchial epithelial BEAS-2B cells were cultured in RPMI 1640 with L-glutamine (Corning, 10-040-CV) supplemented with 10% FBS, 200 mg/mL streptomycin and 200 IU/mL penicillin at 37°C, 5% CO₂. The African green monkey kidney Vero E6 cells were maintained in DMEM (Gibco, 11965092) supplemented with 10% fetal bovine sera (FBS), 200 mg/mL streptomycin, and 200 IU/mL penicillin at 37°C, 5% CO₂. For cell viability assay, the cells were seeded in a 96-well plate with 1 × 10⁴ cells/well and cultured for 24 h, then the supernatants were discarded, and 150 μl of serial-diluted EPSs in culture medium was added to the cells and incubated for another 24 h. Subsequently, 15 μl of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, 5 mg/mL] was added to each well and incubated for 4 h, then the supernatants were discarded and 100 μl of DMSO (dimethyl sulfoxide) was added to dissolve the purple precipitate. Finally, the 96-well plate was scanned with Infinite M200 Pro (TECAN) at 405 nm. Data were expressed as the means ± standard errors of the means (SEM). p values were analyzed by unpaired t test with GraphPad 5.

At each of the study sites, venous blood was collected from consented participants into BD Vacutainer CPT Mononuclear Cell Preparation Tubes (BD Biosciences) with sodium citrate. Tubes with separated blood samples were centrifuged for 10 minutes at 2000 rpm on site, and then stored on ice for ~ 8 hours during transportation to the University of Florida research lab in Gressier, Haiti. Both plasma and PBMCs were then separated via further centrifugation for 10 minutes at 2000 rpm. Plasma was decanted and stored at -80°C. Cells were enumerated using 0.4% trypan blue (Thermo Fisher Scientific) and a hemacytometer (Hausser Scientific) and stored at -80°C in RPMI 1640 with L-glutamine and 25mM HEPES (Corning Mediatech) supplemented with 10% heat-inactivated Hi-FBS (Gibco Life Technologies) and Penicillin/Streptomycin solution (Gibco Life Technologies) with 10% molecular grade DMSO. Samples were shipped on dry ice via air courier to the Emerging Pathogens Institute at the University of Florida, Gainesville, FL. Upon arrival to the Emerging Pathogens Institute, plasma was stored at -80°C and PBMCs were stored in liquid nitrogen until used in experiments.

HEK293T, U2OS, and MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC CRL-3216, HTB-26, and HTB-96, respectively; Manassas, VA, USA) within the previous 24 months and passaged < 10 times for all experiments (passage numbers from 6–15). HEK293T cells were grown in DMEM high glucose plus L-Glutamine supplemented with 1% sodium pyruvate (Hyclone, Logan, UT, USA, #SH30022.01) and 10% fetal bovine serum (FBS, Premium Select, R&D systems, Minneapolis, MN, USA). U2OS cells were grown in RPMI 1640 with L-glutamine (Corning, Glendale, AZ, USA, 10-040-CV) supplemented with 10% FBS. MDA-MB-231 cells were grown in DMEM high glucose plus Glutamax (Life Technologies, Carlsbad, CA, USA, #10566016) supplemented with 1% sodium pyruvate and 10% FBS. Biweekly testing for mycoplasma contamination was performed using the Lonza MycoAlert (Lonza, Bend, OR, USA, #LT07-318).
Low-glucose-adapted HEK293T and MDA-MB-231 cells were grown in DMEM low glucose and pyruvate (Thermo Fisher Scientific, Waltham, MA, USA, #11885084) supplemented with L-glutamine, 1% sodium pyruvate, and 10% FBS. Low-glucose-adapted U2OS cells were grown in RPMI 1640 with L-glutamine without glucose (Life Technologies #11879020), supplemented with 5mM glucose (D-(+)-Glucose Solution Sigma, St. Louis, MO, USA, #G8644) and 10% FBS.