Thp 1 cell

Gallium(III) meso-tetraphenylporphyrin chloride (GaTP) was purchased from Frontier Scientific (Logan, UT). THP-1 cells were purchased from ATCC. Difco Middlebrook 7H9 broth and BBL Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium were purchased from BD (Sparks, MD, USA). 7H10 agar plates were purchased from Remel Inc. (Lenexa, KS). HyClone RPMI 1640, Dulbecco modified Eagle medium (DMEM), l-glutamine, HEPES, sodium pyruvate, and fetal bovine serum were purchased from GE Life Sciences (Logan, UT). Macrophage colony-stimulating factor (MCSF) was purchased from BioLegend (San Diego, CA). Fe-free 7H9 medium was prepared as described previously (11 (link)). Human monocytes that had been purified by countercurrent centrifugal elutriation from normal human donors were purchased from the UNMC Elutriation core facility using an institutional IRB-approved protocol. HIV-1_ADA was obtained from UNMC, Omaha, and its potency was tested by reverse transcriptase (RT) assay before infection. H37Rv was obtained from ATCC and grown in a biosafety level 3 (BSL3) lab, and its potency was tested by counting CFU before infection.

THP-1 cells (ATCC) were grown at 37 °C with 5% CO₂ in RPMI 1640 (Biological Industries, USA) with 10% fetal bovine serum (FBS; Biological Industries, USA). This complete medium is referred to as R10 hereafter.

Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM and with 10% heat-inactivated fetal bovine serum (FBS), 1× penicillin/streptomycin (100×), and 1× l-glutamine (100×) (all from Euroclone, Milano, Italy).
To induce polarization of THP-1 cells into the M0 phenotype macrophages, cells were treated with phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) for 24 h. Afterwards, LPS (15 ng/ml) in RPMI medium with 10% FBS was added for 48 h to polarize M0 macrophages into the M1 phenotype (pro-inflammatory), whereas the M2 phenotype (anti-inflammatory) was induced with IL-4 (25 ng/ml) and IL-13 (25 ng/ml) in RPMI medium with 10% FBS for 48 h. THP-1 cells were maintained in high-glucose medium (60 mM) for 1 week and then treated with 100 ng/ml PMA for 24 h, to mimic hyperglycemia and to obtain macrophages (HgSMs). In order to examine THP-1 proliferation after 1 week treatment with 60 mM d-glucose, cells were plated in 12-well plates at a density of 200,000 cells/ml, treated without or with 60 mM of d-glucose (NG and HG respectively) and then counted after 1 week treatment. The experiment was performed in triplicate.

The breast cancer cell line MDA-MB-231 and MCF7 cells, purchased from ATCC, were maintained and grown in Dulbecco’s modified Eagle’s high glucose (4.5 g/L) medium. This medium was supplemented with fetal bovine serum (FBS; 10%), penicillin-streptomycin (10,000 IU/mL and 10,000 μg/mL), sodium pyruvate (100 mM), non-essential amino acids (1×), and L-glutamine (200 mM). All cells were grown at 37 °C with 90% humidity, 5% CO₂, and 95% air. Kaiso-depleted MDA-MB-231 cells, named sh-Kaiso cells, and Kaiso-scrambled MDA-MB-231 cells, named sh-Scr, were grown in DMEM media using the supplements mentioned here, except that puromycin (0.8 µg/mL) was added in the media to maintain selection. THP1 cells, also purchased from ATCC, were maintained and grown in RPMI-1640 medium with the above-mentioned supplements.

Human monocyte THP-1 cells (ATCC; Rockville, MD, USA) were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin in a humidified incubator at 37 °C and 5% CO₂. Exponential-phase THP-1 monocytes with passage numbers less than 25 were simultaneously stimulated with 100 ng/mL LPS from Escherichia coli (O111:B4) and different concentrations of MSE (4, 8, and 16 μg/mL) for 3 h. The MSE concentrations were selected according to cell viability (>90%) via the MTT assay, as described by Chanput, Reitsma [30 (link)]. The expression of proinflammatory genes TNF-α, IL-1β, IL-6, and IL-8 was measured using qPCR with primer sequences indicated in Chanput, Mes [31 (link)]. Glyceraldehyde-3-phosphate dehydrogenase and the 0 h time point of nonstimulated cells were used for the ΔΔCt normalization. Results were expressed as the relative fold change [31 (link)].