The determination of drug bactericidal activity against nutrient-starved M. abscessus cultures was performed as previously described (52 (link)). Midlog phase M. abscessus Bamboo precultures (OD600 = 0.4–0.6) were spun down at 3200 × g for 10 min at 25°C and washed three times with phosphate-buffered saline containing 0.025% Tyloxapol (Sigma-Aldrich T0307-10G, St. Louis, MO, United States) (PBS/Tyloxapol). Washed culture was then diluted with PBS/Tyloxapol to OD600 = 0.2 (1.8 × 107 CFU/ml). One hundred milliliter of OD600 = 0.2 cultures were incubated in roller bottles (Corning 430195, Oneonta, NY, United States) at 37°C and 2 rpm, and 0 h time point CFU were determined by plating on 7H10 agar. At 144 h post-incubation, CFU were determined again and 1 ml aliquots of the roller bottle cultures were transferred into 14 ml vented, round-bottom tubes (Thermo Fisher 150268, Rochester, NY, United States). Appropriate concentrations of drugs up to 100 μM were added to the tubes prior to incubation at 37°C on a tilted rack in an orbital shaker at 220 rpm. After 48 h of incubation with antibiotics (192 h time point) CFU were enumerated. The MBC was defined as the lowest concentration of drug that reduced the 192 h CFU/ml value by 10-fold relative to the 144 h CFU/ml value.
Tyloxapol
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, France
Tyloxapol is a nonionic surfactant used in laboratory settings. It functions as a dispersing and emulsifying agent. The core purpose of Tyloxapol is to facilitate the suspension and uniform distribution of various materials in aqueous solutions.
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Lab products found in correlation
Glycerol, by Merck Group (16 mentions)
Tween 80, by Merck Group (10 mentions)
Oadc enrichment, by BD (8 mentions)
Cholesterol, by Merck Group (6 mentions)
Middlebrook 7h9 medium, by BD (5 mentions)
Kanamycin, by Merck Group (4 mentions)
Chloroform, by Merck Group (4 mentions)
3h triolein, by PerkinElmer (4 mentions)
Potassium clavulanate cla, by Merck Group (3 mentions)
Sulfatide, by Avanti Polar Lipids (3 mentions)
Endoglycosidase h, by New England Biolabs (3 mentions)
Monoq 10 100 gl, by GE Healthcare (3 mentions)
Acetone, by Merck Group (3 mentions)
Bcg danish, by Statens Serum Institut (3 mentions)
Hybri max, by Merck Group (3 mentions)
Gentamicin, by Merck Group (3 mentions)
Oleic acid albumin dextrose catalase oadc, by BD (3 mentions)
3h cholesterol, by PerkinElmer (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Glycerol, by Thermo Fisher Scientific (3 mentions)
173 protocols using tyloxapol
1
Bactericidal Activity against M. abscessus
2
Purification and Loading of CD1a Glycoprotein
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The glycoprotein CD1a was expressed in a mammalian expression system and purified as previously described9 (link). Following an endoglycosidase H (New England BioLabs) treatment, the purified CD1a was first loaded with the ganglioside GD3 (GD3) (Matreya LLC) that was dissolved in a solution containing 0.5% tyloxapol (Sigma) and 10 mM Tris buffer at pH 8.0. CD1a was first incubated with GD3 overnight at room temperature at a molar ratio of 1:15. The CD1a sample loaded with GD3 was further purified using ion exchange chromatography (MonoQ 10/100 GL-GE Healthcare). Urushiol C15:2 (Chemos) was dissolved in a solution containing 10 mM Tris buffer at pH 8.0 / 0.5% tyloxapol / 50% acetone (Sigma). The GD3–CD1a sample was then incubated overnight with urushiol at a 1:15 molar ratio and at room temperature in order to achieve the GD3 displacement by urushiol. A subsequent purification step involving ion exchange chromatography (MonoQ 10/100 GL) was performed to remove the excess of urushiol C15:2, GD3 –CD1a, and detergent.
3
Culturing Mycobacterium tuberculosis in Defined Media
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Mtb H37Rv (ATCC) was initially grown in Middlebrook 7H9 medium (7H9) supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD, United States), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v; Sigma) prior to inoculation in a MM consisting of KH2PO4 1 g/l, Na2HPO4 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, CaCl2 0.5 mg/l, ZnSO4 0.1 mg/l, with or without Tyloxapol 0.05% (v/v), containing 0.1% (v/v) glycerol, pH 7.0. Cultures were grown in 7H9 at 37°C until they reached an OD of 0.6 and then washed in in phosphate-buffered saline (PBS) + 0.05% Tyloxapol and transferred to MM. Low iron minimal medium (LIMM) was used for culture under iron-controlled conditions. To remove metal ions MM was treated with 5% Chelex-100 (Bio-Rad, Hercules, CA, United States) for 24 h, at 4°C with gentle agitation. Chelex-100 was removed by filtration through 0.22 μm filter (Millipore, Burlington, MA, United States) and the medium was supplemented with sterile ZnCl2 0.5 mg/l, of MnSO4 0.1 mg/l, and of MgSO4 400 mg/l. The amount of residual Fe in this medium, determined by atomic absorption spectroscopy is ∼1 μM. High iron MM (MM) was prepared by supplementing LIMM with 50 μM FeCl3.
4
CD1a Glycoprotein Purification and Ligand Loading
5
Mycobacterial Strains and Culture Conditions
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Mycobacterium bovis strain NEIKER 1403 was originally isolated from a naturally infected wild boar at NEIKER. M. avium subsp. paratuberculosis K10 and M. tuberculosis H37Rv strains were obtained from the ATCC. All strains were initially grown in Middlebrook 7H9 medium (M7H9) supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v; Sigma) prior to inoculation in a minimal medium (MM) consisting of KH2PO4 1 g/l, Na2HPO4 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, MgSO4 × 7 H2O 0.5 g/l, CaCl2 0.5 mg/l, ZnSO4 0.1 mg/l, mycobactin J with or without Tyloxapol 0.05% (v/v), containing 0.1% (v/v) glycerol, pH 7.0. Cultures were grown for up to 14 d for Mtb and 4 weeks for M. bovis and MAP in 850 cm2 roller bottles (Corning) at 37 °C. All EVs preparations were obtained from MM culture supernatants. In some experiments, cultures were Mtb H37Rv cell lysates were generated as previously described [11 (link)].
6
Purification and Loading of CD1a Glycoprotein
7
Exponentially Growing Mab Bamboo Cultures
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Exponentially growing Mab Bamboo precultures (section Growth in Aerated Nutrient-Rich Broth and MIC, MBC90 Determination) were spun down at 3200 × g for 10 min at 25°C and washed three times with phosphate buffered saline containing 0.025% Tyloxapol (Sigma-Aldrich T0307-10G, St. Louis, MO, United States) (PBS/Tyloxapol). Washed culture was then diluted with PBS/Tyloxapol to OD600 = 0.2 (1.8 × 107 CFU/mL). One hundred milliliter of OD600 = 0.2 cultures were incubated in roller bottles (Corning 430195, Oneonta, NY, United States) at 37°C and 2 rpm. CFU were determined by plating. For determination of MBC90 against nutrient-starved non-replicating culture, 1 mL aliquots of 144 h old roller bottle cultures (Figure 1A ) were transferred into 14 mL vented, round-bottom tubes (Thermo Fisher 150268, Rochester, NY, United States) and appropriate concentrations of drugs up to 100 μM were added. The tubes were incubated on a tilted rack at 37°C on an orbital shaker at 220 rpm for 48 h of incubation with antibiotics before plating and CFU enumeration.
8
Mycobacterial Strain Culturing Protocol
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The Mtb H37Rv strain (obtained from C. Sassetti) was grown at 37 °C in a minimal medium (Difco Middlebrook 7H9 broth (BD, 271310) supplemented with 0.5% (v/v) glycerol, 0.05% (v/v) tyloxapol (Sigma, T8761), 0.2 g l−1 casamino acids (BD, 223050), and 10% (v/v) OADC (oleic acid, albumin, dextrose and catalase; BD, 212351)). The double-auxotrophic Mtb mc26206 strain (H37Rv ΔpanCDΔleuCD)42 (link) (obtained from W. Jacobs Jr) was grown in the minimal medium with an additional 50 mg l−1l -leucine (Sigma, L8000) and 24 mg l−1 pantothenic acid (Sigma, P5155). The Msm mc2155 strain (obtained from S. Fortune) was grown in the Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween-80 (VWR, M126), and 10% (v/v) albumin–dextrose–catalase. Liquid Mtb and Msm cultures were grown at 37 °C in Nalgene sterile square PETG medium bottles with constant agitation. The solid Mtb culture was grown on 7H11 agar (Sigma, M0428) supplemented as described above except for tyloxapol.
9
Tyloxapol-Induced Lipid Metabolism Assay
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Tyloxapol was purchased from Sigma-Aldrich (USA); 10% Tyloxapol solution was prepared using saline. Mice were fasted for 6 h and injected with Tyloxapol (500 mg/kg) via the penile vein. Blood was collected from the tail vein before injection (0 min) and at 15, 30, 60, 90, 120, and 180 min after injection. Plasma was separated, and triglyceride and cholesterol concentrations were determined. Triglyceride and cholesterol secretion rates were calculated from the linear regression analysis of time versus concentration.
10
CM Secretion and VLDL Secretion Assays
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For CM secretion, chow diet-fed mice were fasted for 16 h and injected with 500 mg tyloxapol/kg body weight (Merck KGaA; Darmstadt, Germany) to inhibit peripheral lipolysis. Thirty minutes later, mice were gavaged with 200 μl olive oil as a substrate to trigger CM synthesis (Jackson et al., 2002 (link)). Blood was taken prior to the injection as well as 1, 2, 3, and 4 h post olive oil gavage. Plasma TG and TC concentrations were measured as described above. For analysis of CM size, mice were fasted for 4 h prior to injection of tyloxapol (500 mg/kg body weight). One hour later, mice were administered an olive oil bolus (200 μl) and blood was collected 90 min post gavage. Plasma was obtained via centrifugation (7 min at 5,200 x g and 4°C) and pooled for each genotype. Samples (125 μl) were mixed with 280 μl of buffer (PBS, 2 mM benzamidine, 4 M KBr) and 4 mL of 0.9% NaCl before ultracentrifugation for 45 min. CMs were isolated from the upper phase and size was measured in technical triplicate by light scattering (Malvern Zetasizer, Malvern Panalytical GmbH, Kassel, Germany).
VLDL secretion was determined in 16 h-fasted mice after tyloxapol injection (500 mg/kg body weight). Blood was taken prior to injection as well as 1, 2, 4, and 6 h post-injection.
VLDL secretion was determined in 16 h-fasted mice after tyloxapol injection (500 mg/kg body weight). Blood was taken prior to injection as well as 1, 2, 4, and 6 h post-injection.
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