Ultrasonic processor

ChIP assay was performed as previously described with minor modifications (Sunadome et al, 2014). Briefly, cells were cross‐linked by formaldehyde (1%) and quenched by glycine (0.125 M). Pellets were washed and resuspended in SDS lysis buffer with protease and phosphatase inhibitor. Chromatin was sonicated to obtain fragments of about 600 bp (Cole‐Parmer Ultrasonic Processor), incubated with 2 μg rabbit normal IgG or anti‐p‐CREB (Ser133) antibody at 4°C overnight, enriched using protein A+G magnetic beads, washed by low‐salt wash buffer, high‐salt wash buffer, and LiCl wash buffer, and released using elution buffer at 65°C. DNA was treated with RNaseA (0.2 mg/ml) and proteinase K (0.2 mg/ml) and purified as template for quantitative PCR to amplify CREB‐binding region in the FST promoter region. c‐Fos was used as positive control of p‐CREB ChIP‐qPCR. Compositions of various buffers and reagents, including dilution of each antibody, are shown in Table EV5.

PLGA NPs containing either RIF or INHP were prepared by double emulsification using sonication. Briefly, PLGA was dissolved in HPLC-grade dichloromethane (DCM). The drug was then added to the DCM/PLGA solution and mixed to obtain complete dissolution. This solution was added to 1% polyvinyl alcohol (PVA) cooled in an ice bath and sonicated using an ultrasonic processor (Cole Parmer, Vernon Hills, IL, USA) at 20% amplitude for 10 min. Particle size, polydispersity index (PDI), and surface charge (ζ potential) were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano Series Nano-ZS (Malvern Instruments, Inc., Westborough, MA, USA). The suspension was mixed overnight at room temperature to evaporate DCM and then collected after 24 h and centrifuged stepwise to 8000 g at 5°C for 20 min. After the supernatant was decanted, the pellet was washed twice in 25 ml of deionized water by centrifugation at 8000 g for 20 min. The particle size was determined by DLS, and drug concentrations were determined by reversed-phase high-performance liquid chromatography (HPLC) with UV/Vis detection (37 , 38 ).

The strains M. microti-pMV261, M. microti-PstS-1 and M. microti-Ag85 were grown in 250 mL flasks containing 50 mL of Sauton media (composition per liter: 4 g l-asparagine, 0.5 g monopotassium phosphate, 0.5 g magnesium sulphate, 50 mg ferric ammonium citrate, 2 g citric acid, 60 mL glycerol and 0.05% Tween 80), supplemented with 40 mM of glucose and 40 mM of sodium pyruvate at 37 °C with constant stirring at 150 rpm. The kinetic growth was determined every 48 h for 196 h by measuring the O.D. (600 nm) and expressed in absorbance units (A.U.). At the end of the culture, 1 mM of PMSF (phenylmethylsulphonyl fluoride) as a protease inhibitor, 100 µM of EDTA and 0.05% sodium azide were added to the supernatant. The supernatant was concentrated to a volume of 10 mL using an Amicon^® Stirred Cell (Merck™) system and ultrafiltration discs with a nominal molecular weight of 3 kDa (Merck™). It was then filtered again using an Amicon^® Ultra-15 Centrifugal Filter Unit (Merck™). The proteins from the cell extract were recovered by sonicating the centrifuged bacterial pellet with 15 pulses of 100 mV (1 min on/1 min off) at 4 °C in an Ultrasonic Processor (Cole-Parmer^®). To prevent proteolysis, 1 mM of PMSF was added during the sonication. Finally, the concentrated supernatants and cellular extracts of the three strains were recovered and stored at −70 °C until further use.

For RNA extraction, the tissues were cut into small pieces with microscissors and lysed in RNA isolation reagent (RNA-Bee, Tel-Test Inc., Friendswood, TX, USA). Then, the tissues were homogenized with an ultrasound sonicator (Ultrasonic Processor, Cole Parmer Instruments, Vernon Hills, IL, USA). Total RNA was extracted with an RNeasy Mini kit (Qiagen, Valencia, CA, USA) and was converted to the first-strand cDNA by reverse transcription using High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). The cDNA was analyzed by real-time PCR amplification for TNF-α and IL-1β on an ABI 7500 Real Time PCR System (Applied Biosystems). All probe sets were purchased from Applied Biosystems (TaqMan Gene Expression Assay kits). Mouse-specific GAPDH was used as internal controls.

T-ALZ01 was loaded into exosomes through sonication. T-ALZ01 (0.2 mg) was added to 200 μL of exosomes (~ 1×10⁸ exosomes) and the mixture was sonicated using a Cole-Parmer Ultrasonic Processor (Vernon Hills, IL, USA). The sonication settings used were 500 v, 2 kHz, 20% power, 6 cycles by 4 seconds pulse and 2 seconds pause according to Haney et al. [38 (link)]. The mixture was allowed to cool on ice for 2 minutes, and then the sonication cycle was repeated. The success of the drug loading was analyzed using high performance liquid chromatography (HPLC).