48 well plate

For phenotyping, macrophages were stained with anti-CD14-PE and anti-CD163-allophycocyanin in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), 2% FBS, 1% pooled human serum (Lonza) and 0.1% sodium azide. For CD14 and CD163 expression analysis after co-stimulation, cells were harvested using TrypLE Select (Invitrogen) and stimulated in 48-well plates (Greiner Bio-one) for 24 h in IMDM without phenol red (Gibco). Fluorescence of the cells was assessed by flow cytometry (Canto II, BD Biosicences).

PBMCs were isolated from whole blood, of healthy TST negative donors (n = 12), using Ficoll-Paque PLUS (GE Healthcare Life Sciences, MA, United States) density (D > 1.077 g/ml) gradient centrifugation. Informed consent was obtained from all the subjects and the study was approved by the Ethical Review Committee of the Faculty of Health Sciences at Stellenbosch University (N16/05/070). Cells were cultured in Roswell Park Memorial Institute (RPMI) media, supplemented with 10% FBS at a density of 5 × 10⁵ cells per well in 48-well plates (Greiner Bio-one, Kremsmünster, Austria). PBMCs were then infected with M. tuberculosis H37Rv or M. tuberculosisΔleuDΔpanCD at an MOI of 10:1, treated with penicillin/streptomycin, followed by washing as described above, before adding fresh RPMI, containing 10% FBS. Uninfected and Lipopolysaccharide- (LPS; 10 μg/ml) stimulated cells were included as negative and positive controls, respectively. Supernatants were collected 24 h post-infection and stored at -80°C until cytokine analysis. To assess uptake, PBMCs were lysed with sterile distilled water and pipetting, followed by serial dilution plating and CFU determination as described above.

To assess cell metabolic activity, MC3T3-E1 pre-osteoblasts were seeded on OCP-coated titanium discs without/with κ-carrageenan at 1×10⁵ cells/disc in 48-well plates (Greiner), and cultured up to 7 days. The medium was removed, cells were washed with PBS, and α-MEM with 10% FBS and antibiotics was added. The PrestoBlue™ Assay (Invitrogen) was used to evaluate cell metabolic activity according to the manufacturer’s instructions. In short, PrestoBlue™ reagent was added to the cells (10%, vol/vol), followed by 30 min incubation in a 5% CO₂ in air incubator with a humidified atmosphere at 37°C. The medium was harvested (100 μl/well) and transferred into a 96-wells black microplate (Greiner). Fluorescence intensity was determined at a wavelength of 560 nm (excitation) and 590 nm (emission), and quantified using a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific). Prestoblue™ fluorescence was linearly associated with DNA content (data not shown).

HT-29 cells were adjusted to a density of 5 × 10⁵ cells/mL and 1 mL cell suspension was seeded into 24-well plates (TPP Techno Plastic Products AG, Trasadingen, Schweiz) and incubated for adherence overnight. Jurkat cells were adjusted to a density of 2.5 × 10⁵ cells/mL and 1 mL was seeded into 48-well plates (Greiner bio-one, Frickenhausen, Germany). Then, cells were pre-incubated with free hypericin for 16 h in the dark (plates were wrapped with aluminum foil) to enable uptake by the cells. Methanol-treated cells and phosphate buffered saline (PBS)-treated cells served as controls. Then, cells were illuminated with a slimlite light-emitting diode (LED) white light source (40 W/m², Kaiser, Buchen, Germany) for various time intervals to start the phototoxic reaction of hypericin.

Evaluation of cholesterol efflux capacity was performed as described elsewhere [40 (link),41 (link)]. J774.2 cells (Sigma-Aldrich, Darmstadt, Germany) were cultured in DMEM (Life Technologies, Carlsbad, CA, USA), containing 10% fetal bovine serum and 1% penicillin/streptomycin. In each well, 300,000 cells were seeded on 48-well plates (Greiner Bio-One, Kremsmünster, Austria), cultured for 24 h and labeled with 0.5 µCi/mL radiolabeled [³H]-cholesterol (ART0255, Hartmann Analytic, Braunschweig, Germany) in DMEM containing 2% BSA, in the presence of 0.3 mM 8-(4-chlorophenylthio)-cyclic adenosine monophosphate (c3912, Sigma-Aldrich, Darmstadt, Germany) overnight. Cyclic adenosine monophosphate was used for the upregulation of ATP-binding cassette transporter A1. After 18 h, the cells were rinsed with DMEM (serum-free) and equilibrated with DMEM (serum-free) containing 2 mg/mL bovine serum albumin (Sigma-Aldrich, Darmstadt, Germany) for 2 h. Cells were then incubated with 2.8% apoB-depleted plasma for 3 h to determine [³H]-cholesterol efflux. The cholesterol efflux capacity was expressed as the radioactivity in the medium in relation to the total radioactivity in medium and cells. All steps were performed in the presence of 2 µg/mL acyl-coenzyme A cholesterol acyltransferase inhibitor Sandoz 58-035 (Sigma-Aldrich, Darmstadt, Germany).

HCjE-Gi cells were cultured in 48-well plates (Greiner). On culture days 3, 5, 7, 14, 21, and 28, the cell-secreted ECM was isolated. Following thorough PBS washes, fresh HCjE-Gi cells were seeded onto the cell-deposited ECMs at a density of 1 × 10⁴ cells/cm² in KSF culture media. Tissue culture polystyrene (TCP) incubated with 1% NH₄OH solution and then thoroughly washed with sterile PBS was used as the control substrate. We then assessed the cell numbers on the different culture conditions on culture days 1, 3, 5, and 7 after seeding. The cells were fixed with 100% ice-cold methanol for 2 minutes, air dried, and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies; 5 mg/mL diluted 1:30,000 in PBS) for 5 minutes at room temperature (RT). Images were acquired with a 10× objective from five fixed positions of each well, and the number of nuclei per view was determined. The number of nuclei per view on cell-secreted ECM was divided by the number of nuclei of the corresponding view on TCP to obtain ECM:TCP cell number ratios. All experiments were carried out three times, with three technical repeats per independent experiment.