50 μg/ml Hygromycin (Invitrogen), 20 μg/ml Kanamycin (GoldBio), 12.5 μg/ml zeocin (Invitrogen), 0.2% acetamide (Sigma), and 50 ng/ml of anhydrous tetracycline (ATc) (Sigma) were used unless otherwise indicated. H2O2, MMS, streptomycin, isoniazid, rifampicin, ciprofloxacin (all from Sigma) were used at the indicated concentrations.
Anhydrous tetracycline
Manufactured by Merck Group
Anhydrous tetracycline is a type of laboratory equipment used for the storage and handling of the antibiotic compound tetracycline in its anhydrous form. It is designed to maintain the stability and purity of the compound in controlled environments.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Merck Group (2 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Rifampicin, by Merck Group (1 mentions)
Isoniazid, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Kanamycin, by GoldBio (1 mentions)
Acetamide, by Merck Group (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
4 protocols using anhydrous tetracycline
1
Antibiotic Sensitivity Profiling
2
Chlamydia trachomatis Infection of HeLa Cells
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HeLa cells (ATCC) at 80–95% confluence in a 6- or 12-well cluster plate were inoculated with C. trachomatis diluted in Hank’s Balanced Salt Solution prior to centrifugation (500xg for 15 minutes at 20°C) and incubation at 37°C for 30 minutes. The inoculum was then aspirated before addition DMEM supplemented with 10% fetal bovine serum (FBS) and other supplements as noted. For cassette induction, anhydrous tetracycline (Sigma) was supplemented to a final concentration of 5 ng/ml at 6 hours post infection.
3
Cloning LLO into Listeria strain
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LLO cDNA was cloned into the pPL2 plasmid and introduced into the hly KO Listeria strain DP-L2161 by conjugation with E. coli SM10. Following the selection of the bacteria on BHI agar plates with 50 μg/ml streptomycin (Sigma-Aldrich, S9137) and 15 μg/ml chloramphenicol (Sigma-Aldrich, C0378), they were grown overnight in the presence of 100 ng/ml anhydrous tetracycline (Sigma-Aldrich, 37919). The pPL2 plasmid and E. coli SM10 were kindly provided by Dr Anat Herskovits.
4
Metabolic Analysis of Leucine and BCAA in Cells
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For analysis of leucine or other BCAA metabolism, cells were inoculated in 4 mL of 2xYT containing appropriate antibiotics in a 14-mL round bottom tube and incubated at 37 °C with shaking at 250 RPM overnight. The next day, cell cultures were diluted 1:100 in 50 mL of FM1 medium in 125-mL baffled flasks at 37 °C with shaking (250 RPM) for 2h, also with appropriate antibiotics. Cells were induced with the addition of anhydrous tetracycline (Sigma, ATC, 100 ng/mL final concentration) or IPTG (Sigma, 1 mM final concentration). All induction proceeded for 4h. Following induction, cells were centrifuged for 5 minutes at 8000 x g at 4 °C, washed once with an ice-cold formulation buffer (KH2PO4 (2.28 g/L) and K2HPO4 (14.5 g/L)) containing 15% of glycerol (pH 7.5).
Finally, cells were concentrated 20-fold in the formulation buffer and stored at 80°C until the days of testing.
Finally, cells were concentrated 20-fold in the formulation buffer and stored at 80°C until the days of testing.
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