Phosphate buffered saline pbs

Overnight cultures of DH10B cells transformed with FRET substrate expression vectors were diluted 1:50 and grown at 37°C for 3 h in LB medium (Sigma) supplemented with 34 μg/ml chloramphenicol. Expression of FRET proteins was induced with 0.1% wt/vol L(+)-arabinose (Sigma) for 16 h at room temperature. Cells were harvested by centrifugation and soluble protein was then isolated using B-PER Protein Extraction Reagent (Pierce). Fusion proteins, which contain a C-terminal 6×His tag, were then purified using HisPur Ni-NTA resin (Pierce) and eluted in Phosphate Buffered Saline (PBS, Quality Biological), pH 7.4 with 150 mM imidazole (Sigma).

Mycobacterium tuberculosis strain CDC1551 (Ahmad et al., 2009 (link)) was used in all experiments. Prior to exposure to stress conditions, bacterial cultures were grown from frozen stocks to early exponential-phase (OD_600nm = 0.3) in Middlebrook 7H9 broth supplemented with 10% OADC enrichment (BD), 0.2% glycerol, and 0.05% Tween-80.
For nutrient starvation studies, the early exponential-phase bacteria were washed twice with phosphate-buffered saline (PBS) (Quality Biological Gaithersburg, MD) and resuspended in 100 ml of PBS containing 0.05% Tween-80 in a 250-ml flask. The final OD_600nm after resuspension was approximately 0.1. Cultures were incubated at 37°C for 3 days without shaking prior to RNA extraction. For progressive hypoxia studies, early exponential-phase bacteria were washed twice with Dubos-Tween-Albumin broth (DTA) and then resuspended in DTA containing 0.5 μg/mL methylene blue to a final OD_600nm of approximately 0.001. Twenty milliliters of this culture was added to 30-mL cylindrical glass tubes (19 × 145 mm) each containing a magnetic stir bar and plugged with a rubber stopper to prevent gas exchange. The tubes were then placed upright on a magnetic platform and checked every 2–3 days for color changes. Samples were collected when tubes were observed to have a yellowish color (about 14 days after inoculation).

Trephined corneal tissue was placed and secured on an artificial anterior chamber (Katena, Parsippany, NJ, USA) (Supplementary Fig. 1, 1A). Using 0.12 mm Colibri forceps (Storz Ophthalmic Instruments/Bausch & Lomb Corporation, Vaughan, CA), the conjunctival tissue overlying the limbal area (Supplementary Fig. 1, 1B, asterisk) was reflected to expose the limbus (Supplementary Fig. 1, 1B, arrowhead). 10-µl trypan blue or 10-µl black India ink was injected into the limbus by using a beveled 34-gauge needle (JBP Nanoneedle, South Korea) and a 1-ml syringe (BD Syringe, Franklin Lakes, NJ, USA) (Supplementary Fig. 1, 1C,D). The corneal tissue was then transferred to a humidified environment in a sterile 6-well cell culture plate containing one sheet of folded 2″ × 2″ sterile gauze soaked in 2 ml of phosphate-buffered saline (PBS; Quality Biological, Gaithersburg, MD, USA). It was left undisturbed at room temperature for 4 h before undergoing histological processing (see Histology below). Supplementary Video 1 demonstrates an exemplary video of trypan blue injection.