1n naoh

Collagen was extracted and reconstituted as previously described [51 (link),52 (link)]. At the time of fabrication, stock collagen solution was returned to pH 7.0 and maintained at 300 mOsm by mixing with appropriate volumes of 1N NaOH (Sigma-Aldrich, St. Louis, MO), 10x PBS, and 1x PBS as previously described [26 (link)]. Collagen solution was immediately mixed with cells suspended in PBS and formed into disc constructs as previously described [17 (link),23 (link)]. Briefly, cell suspensions were formed with AuC:MSC ratios of 1:0, 1:1, and 0:1. Neutralized collagen was then homogeneously mixed with the cell suspensions for a final cell concentration of 25 x 10⁶ cells/mL and collagen density of 10 mg/mL. The collagen hydrogel was extruded between two glass plates spaced 2 mm apart and allowed to undergo thermal gelation at 37 °C for 1 hour. Following gelation, 8 mm diameter disc constructs were formed using a dermal biopsy punch and placed in the same media used for cell expansion. Constructs were implanted within 48 hours.

Melanin content was determined by the method of Buscà et al. [24 (link)] with a few modifications. The cell pellets were dissolved in 0.5 mL of 1 N NaOH at 100°C for 30 min, then determined cell counts, and transferred 30 μL to the well of 96-well plate. The 170 μL of L-DOPA (0.001 g/mL) was mixed to each well to incubate together for 5 min. The melanin content was measured by the optical absorbance at 450 nm and compared with a standard curve generated by melanin with known concentrations in 1 N NaOH (Sigma Chemical Co., St. Louis, MO). The amount of melanin was further normalized by cell counts obtained previously and expressed as g melanin/cell. Each sample was measured in duplicate.

Davisco Foods International (Le Sueur, MN, USA) and Cremer (Hamburg, Germany) kindly donated whey protein isolates BiPro^® and Miglyol 812 N^®, respectively. The whey protein comprised at least 97% of dry basis protein, mainly β-lactoglobulin and α-lactalbumin protein. Miglyol 812 N^® was kindly donated by Cremer (Hamburg, Germany). Hyaluronic acid sodium (from Streptococcus equi), maize amylopectin, alginic acid sodium, and griseofulvin (from Penicillium griseofulvin—97.0–102.0%) were purchased from Sigma-Aldrich. KCl and citric acid anhydrous were also purchased from Sigma-Aldrich. NaH₂PO₄ and Na₂HPO₄ anhydrous, 1 N HCl, 1 N NaOH, and MeOH were purchased from Sigma-Aldrich (Kempton Park, South Africa). Acetonitrile, LiChrosolv^®, was acquired from Merck (Kempton Park, South Africa).

Deionized water, methanol (99.9%), and isopropanol (99.9%) were obtained from Fisher Scientific (Waltham, MA). 1 N NaOH, 1 N HCl, 5 N ammonium hydroxide, glacial acetic acid (99.99%), ultra-high-purity ammonium acetate (99.999%), total human serum IgG isolate (purity ≥95%, based on non-reduced SDS-PAGE and verified by nanoLC-MS/MS of tryptic digests), and BSF isolate (purity ≥90%) were purchased from Sigma-Aldrich (St. Louis, MO). Agencourt Cleanseq carboxyl-coated magnetic microparticles (COOH-beads) were from SCIEX (Brea, CA). PNGase F enzyme and bovine pancreas ribonuclease B isolate (purity ≥85%, based on SDS-PAGE) were from New England Biolabs (Ipswich, MA). A neodymium magnet was obtained from K&J Magnetics (Pipersville, PA). All bare-fused silica (BFS) capillaries (91 cm × 30 µm i.d. × 150 µm o.d.) with sheathless CESI-MS emitters in OptiMS cartridges were from SCIEX. A NanoBooster^TM unit for generating the DEN-gas was from Bruker Daltonics (Billerica, MA).

A collagen suspension (5 ml) containing 3.0 ml collagen G1 (5 mg/ml, Matrix biosciences, Morlenbach, Germany), 0.5 ml 10x M199 medium (Sigma), 85 ul 1N NaOH (Sigma) and sterile water was mixed with 1.0 ml (2 × 10⁶ cells) hPSCs. Collagen gel-cells suspension (0.6 ml/well) was plated in a 24-well culture plate and allowed to set for 1 h at 37 °C. Once set, gels were detached from the culture wells and 1 ml of serum free medium was added with or without TGF-β (5 ng/ml). To study the effect of FGF2 and FGF2-SPIONs, 1 ml of serum free medium with TGFβ (5 ng/ml) and FGF2 or FGF2-SPIONs (equivalent to 250 ng/ml FGF2) was added to the detached gels. Representative images were made at 96 h using a normal digital camera. Measurement of collagen gel diameter was performed using ImageJ (NIH).

2.5 × 10⁶ infected PBMCs (250 μl) per well were distributed across 24-well plates. Thereafter, cells were embedded within an extracellular matrix (ECM) by adding 250 μl per well of a mixture composed of 950 μl/ml of collagen (3.1 mg/ml; PureCol collagen solution, Advanced BioMatrix), 50 μl/ml of PBS 10X (Sigma-Aldrich), 4 µl/ml of fibronectin (1 mg/ml; Sigma-Aldrich) and 10 μl of 1N NaOH (Sigma-Aldrich). The ECM was left to set for 45 min at 37°C (5% CO₂) before 500 μl of cell culture medium were added.
Plates were incubated at 37°C (5% CO₂) and granuloma formation was monitored on days 1, 4 and 8 post-infection using a Leica DM IL LED inverted microscope and a Leica MC170 HD camera. The areas encompassing cellular aggregates were scored in ImageJ 1.52n (National Institutes of Health).
On day 4 post-infection, if applicable, 250 μl of supernatant were replaced by the same volume of fresh cell culture medium containing adalimumab (Humira, Abvie) – a humanized anti-human TNF-α antibody – or a relevant isotype control (human IgG1, clone ET901; Biolegend) at a final concentration of 10 ng/ml.
To study the impact of GM-CSF on bacterial load, 2D granulomas were treated with recombinant human GM-CSF (Peprotech) at a final concentration of 5 ng/ml two hours post-infection.

The human, in-vitro granuloma model developed by Kolattukudy and colleagues [28 (link)] was adapted. Briefly, rested PBMCs were infected with Mtb at a multiplicity of infection (MOI) of 0.05 bacteria per monocyte, assuming 10% monocytes in PBMCs, and distributed in 24-well plates at 2.5×10⁶ PBMCs/well. An extracellular matrix (ECM) was prepared by mixing thoroughly 0.95 ml of PureCol collagen solution (Advanced BioMatrix), 50 μl of 10×DPBS (SAFC Biosciences), 4 μl of fibronectin (Sigma), and 10 μl of 1N NaOH (Sigma) per ml of ECM required and kept at 4°C. The ECM solution was mixed with the infected PBMCs in a 1:1 ratio (v/v) at room temperature (RT), and was allowed to set for 45 min at 37°C (5% CO₂). Once the ECM completely set, wells were topped up with 500 μl of cell culture medium and incubated at 37°C (5% CO₂). On day 4 post-infection, when relevant, supernatant was replaced by the same amount of fresh cell culture medium containing the studied antibodies, biologics or the isotype control. Granuloma formation was monitored on day 7–8 post-infection using a Leica DM IL LED inverted microscope and a Leica MC170 HD camera (Leica).