In the ICG uptake assay, bioprinted structures or cells were cultured for 48 hours in a medium supplemented with 8-bromo-cAMP, pregnenolone-16α-carbonitrile, and progesterone, as per the manufacturer's instructions. Subsequently, they were coincubated with ICG (1 mg/ml) at 37°C for 1 hour, washed three times with PBS, and then switched to HEM culture medium. The uptake/release of ICG was observed under a microscope. Bioprinted structures or cells were stained with ac-LDL (Yeasen) and periodic acid-Schiff (Solarbio), and the staining results were observed under a microscope. Different inducers were used to treat 3DP-liver and eHep cells for 48 hours, and the gene expression levels of Cyp1a2 (3-methylcholanthracene, 10 μM, Sigma-Aldrich), Cyp3a11 (rifampicin, 25 μM, Solarbio), Cyp2e1 (rifampicin, 25 μM, Solarbio and ethyl alcohol; 0.5%, v/v), Cyp3a41b (3-methylcholanthracene, 10 μM, Sigma-Aldrich), and Cyp1a1 (rifampicin, 25 μM, Solarbio and barbituric acid, 10 μM, Aladdin) were detected. The P450-Glo Cyp1a2 Induction/Inhibition Assay (Promega) was used, following the manufacturer's instructions, to detect Cyp1a2 enzyme activity in different states.
Rifampicin
Manufactured by Solarbio
Sourced in China
Rifampicin is a chemical compound commonly used as a laboratory reagent. It is a broad-spectrum antibiotic that inhibits bacterial RNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin, by Solarbio (2 mentions)
Sodium azide, by Avantor (2 mentions)
Ampicillin, by Solarbio (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Chloramphenicol, by Solarbio (1 mentions)
Kanamycin, by Solarbio (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Clonexpress 2 multis one step cloning kit, by Vazyme (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Lb nutrient agar, by Solarbio (1 mentions)
Lysogeny broth lb, by Solarbio (1 mentions)
3 methylcholanthracene, by Merck Group (1 mentions)
P450 glo cyp1a2 induction inhibition assay, by Promega (1 mentions)
Periodic acid schiff, by Solarbio (1 mentions)
Acldl, by Yeasen (1 mentions)
Diclofenac sodium, by Merck Group (1 mentions)
Tranylcypromine, by Merck Group (1 mentions)
6 protocols using rifampicin
1
Hepatic Cytochrome P450 Induction Assay
2
Quantifying PA14 Colonization in Worms
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The quantification procedure of intestinal PA14 colony forming units (CFU) was modified from a previous study (39 (link)). Briefly, after exposure to P. aeruginosa PA14 for 24 hours, ten worms were transferred onto a fresh 3.5 cm NGM plate with M9 solution containing 25 mM sodium azide (Amresco, 0639) to paralyze the worms. Worms were then washed once by M9 solution containing 1 mg/ml ampicillin and 1 mg/ml gentamicin (Solarbio, G8170, China) and incubated for 0.5 h to kill bacteria attached to the surface of the animals on the plate. After worms were lysed with a motorized pestle, serial dilutions of the lysates (10-1, 10-2, 10-3, 10-4) were plated onto Luria-Bertani plates containing 100 g ml-1 rifampicin (Solarbio, R8011) and incubated overnight at 37°C to select for PA14 colony. PA14 colonies were counted to determine CFU per worm. At least three independent replicates were performed for each condition.
3
Molecular cloning techniques
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Isopropyl-β-d -thiogalactopyranoside (IPTG) was supplied by Sinopharm Chemical Reagent (Tianjin, China). Chloramphenicol, kanamycin, and rifampicin were supplied by Solarbio Science and Technology (Beijing, China). Taq DNA polymerase and Phusion high-fidelity DNA polymerase were obtained from Thermo Fisher Scientific Inc. (MA, USA). ClonExpress II/MultiS One Step Cloning Kit was purchased from Vazyme Biotech (Nanjing, China). Wizard Genomic DNA Purification Kit was obtained from Promega Corporation (WI, USA). Restriction endonuclease BsaI, T4 PNK, and T4 DNA ligase were purchased from New England Biolabs Inc. (MA, USA). All other chemicals used were of analytical grade or better.
4
Bacterial Conjugation: E. coli Plasmid Transfer
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Plasmid conjugal transfer experiments were carried out with E. coli Ec600 as the recipient and strain Ec15103 as the donor. The recipient and donor bacteria were inoculated in Lysogeny Broth (LB, Solarbio Science & Technology, Beijing, China) and incubated at 37°C and 200 rpm overnight. A total of 50 μL of the overnight cultured bacterial suspension was inoculated in 5 mL of LB broth at 37°C and 200 rpm followed by growth to OD600≈1.0, and then 500 μL of donor and recipient bacteria were mixed. The mixture was spotted onto a 1-cm2 filter membrane placed on LB nutrient agar (Solarbio Science & Technology, Beijing, China). The plates were incubated at 37°C for 12∼18 h, then the cells were washed from the filter membrane to 1 mL of LB broth. After 1,000-fold dilution, 100 μL was spread on LB nutrient agar medium containing 100 μg/mL ampicillin (Solarbio Science & Technology, Beijing, China) and 750 μg/mL rifampicin (Solarbio Science & Technology, Beijing, China). The colonies after overnight culture at 37°C were transconjugants (Ec15103-Ec600), as verified by PCR.
5
Quantifying Pseudomonas Infection in Worms
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The worms were exposed to P. aeruginosa PA14 as described above. After infection for 24 h, worms were transferred to M9 solution containing 25 mM sodium azide (Amresco, 0639) to paralyze the worms and stop the pharyngeal pumping. Worms were washed three times with an antibiotic M9 solution containing 1 mg/ml ampicillin and 1 mg/ml gentamicin (Solarbio, G8170, China) followed by 30 min of incubation in the antibiotic solution to kill the bacteria present on the exterior of the worms. After transfer onto the new NGM plates containing 1 mg/ml ampicillin and 1 mg/ml gentamicin for another 30 min to further eliminate the external P. aeruginosa PA14, the worms were lysed with a motorized pestle. Lysates were serially diluted with M9 buffer and plated on Luria-Bertani plates containing 100 μg/ml rifampicin (Solarbio, R8011, China) to select for P. aeruginosa PA14. After overnight incubation at 37 °C, colonies of P. aeruginosa PA14 were counted to determine CFU per worm. Ten worms were examined per treatment, and at least three replicate assays were performed.
6
Pharmacological Evaluation of Drug Interactions
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Amodiaquine, diclofenac sodium, dextromethorphan, furafylline, tranylcypromine, ticlopidine, montelukast, sulfaphenazole, ketoconazole, omeprazole, atenolol, digoxin (European Pharmacopoeia reference standard), and verapamil were purchased from Sigma-Aldrich. Rifampicin and phenobarbital were purchased from Solarbio, China. Phenacetin, bupropion, flumazenil and minoxidil were purchased from the National Institutes for Food and Drug Control (NIDFC), China. Midazolam was procured from Cerilliant, TX, USA. Mephenytoin and N-3-benzylnirvanol were purchased from Toronto Research Chemicals (TRC), ON, Canada. Quinidine was procured from TCI, Japan. Testosterone (#A0256659, Acros) and acetonitrile were received from Fischer Scientific. ZB716 was synthesized as described in our earlier report [11 (link)].
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