Rpmi 1640 culture medium

The human monocytic leukaemia cell line THP-1 (American Type Culture Collection, Rockville, MD) was grown in RPMI 1640 culture medium (Sigma, Neustadt, Germany) supplemented with 10% (v/v) FBS (Invitrogen, UK) and 1% (v/v) penicillin/streptomycin (P/S) (Invitrogen) at 37 °C in 5% CO₂ humidified incubator. Cells were sub-cultured at 72 h intervals. The macrophage-like cells were obtained by treating 1 mL of 10⁶ THP-1 monocytes/well with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma, Neustadt, Germany) for 48 h in a 12-wells cell culture plate (Greiner, Frankenhauser, Germany). Differentiated and plastic-adherent macrophage-like cells were then washed twice with RPMI culture medium and rested for another 24 h with culture medium to obtain the resting state of macrophages.

Chemicals, such as Ho(NO₃)₃∙5H₂O (99.9%), NaOH (>99.9%), triethylene glycol (TEG; 99%), PEI [50 wt.% in water, M_n = 1200 amu (M_w = 1300 amu; PEI1200) and 60,000 amu (M_w = 750,000 amu; PEI60000)], dimethyl sulfoxide (DMSO) (99.9%), and Rosewell Park Memorial Institute (RPMI)1640 culture medium were obtained from Sigma-Aldrich (Burlington, MA, USA) and used as-received. Ethanol (99.5%) was purchased from Duksan (Ansan, South Korea) and used as-received for the initial washing of nanoparticles. Triple-distilled water was used for the final washing of nanoparticles and for preparing nanoparticle suspension samples (~30 mM Ho).

Human PMNs were freshly isolated from sodium citrate (3.2%) blood of healthy donors. After dextran-sedimentation, density gradient centrifugation using Ficoll-Paque Plus (Sigma-Aldrich) was performed according to the manufacturer’s protocol. Subsequently, hypotonic lysis of remaining erythrocytes was performed by a 20-s incubation in sterile water and stopped by adding an equal volume of 1.8% sodium chloride solution. As the last step, PMNs were resuspended in RPMI-1640 culture medium (Sigma-Aldrich) at a final concentration of 1 × 10⁶ cells/ml and directly used for the experiments.

The cells were inoculated in 96-well plates at a density of 5 × 10⁴/mL. 100 μL cell suspension was added to each well. Blank pores are free of cells. Incubate overnight in a 5% CO₂ incubator. After the cells were completely attached to the wall, the supernatant was carefully sucked from the 96-well plate. In the experimental group, the RPMI-1640 culture medium containing different concentrations of sufentanil (1, 10 μmol/L) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well, 200 μL. The negative control group and the blank control group only added the culture medium 200 μL. Six parallel control holes were set for each group. After incubation for 48 h, 5 mg/mL MTT 20 μL was added to each well for 4 h. Carefully suck off the supernatant. DMSO 150 μL (Sigma-Aldrich, St. Louis, MO, USA) is added per well. Shake the shaker gently for 10 min. After the purple particle was fully dissolved, the wavelength of the purple particle was determined at 570 nm by a microplate reader (MultiskanEX, Lab systems, Helsinki, Finland). Determine the absorbance (OD) value of each well.

NIH:OVCAR-3 cell line was grown in RPMI-1640 culture medium (Sigma-Aldrich, Germany), supplemented with 2 mM L-glutamine, 100 U/mL penicillin/100 µg/mL streptomycin (Sigma-Aldrich), 15% fetal bovine serum (FBS) (Sigma-Aldrich, Germany), and 0.01 mg/mL bovine insulin (Sigma-Aldrich). A2780 and A2780 Cis cell lines were cultivated in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, 1% antibiotics (penicillin + streptomycin) (all reagents from Sigma Aldrich) and with 1 µM cisplatinum in the case of A2780 Cis cell line. Ishikawa cell line was cultivated in Eagle’s Minimal Essential Medium (MEM) supplemented with 10% FBS, 2 mM L-glutamin, 1 mM sodium pyruvate, 1% NEA, and 1% antibiotics [25 (link)]. BJ HEP cell line was cultivated in Eagle’s Minimal Essential Medium (MEM), supplemented with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, 1% antibiotics (penicillin + streptomycin), and 1% Non-Essential Aminoacids (NEA) Solution (Sigma Aldrich) [26 (link)]. HUVEC cell line was cultivated in RPMI medium, with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-glutamine, and 1% antibiotics (penicillin + streptomycin) (all reagents from Sigma Aldrich). Cell cultures in the 12th passage were used [27 (link)].

Peripheral blood mononuclear cells from 18 child patients with active CD on a gluten-containing diet were isolated from 6 mL of heparinized blood by Histopaque gradient centrifugation and cultured at a density of 1 × 10⁶ cells per milliliter in 96-multiwell culture plates in RPMI-1640 culture medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (GIBCO-Invitrogen Ltd), 1% penicillin-streptomycin, and 0.1% gentamicin (Sigma-Aldrich). After 48 h, PBMCs were incubated with different peptides (50 µg/mL). After 48 h of stimulation, the free supernatants were collected and stored at −80 °C until the interferon gamma (IFN-γ) analyses were carried out.