Mice were subjected to CUMS for a total duration of 6 weeks (Figure 1 ). With the exception of those in the control group, all animals were subjected to the mild stress protocol in an unpredictable manner for 6 weeks. The protocol consisted of seven stressors: food deprivation for 24 h, water deprivation for 24 h, restraint stress for 5 h, overnight illumination for 8 h, horizontal oscillation for 20 min, cage tilting at 45° for 24 h, and a soiled cage environment (500 mL water added to 250 g sawdust bedding) for 24 h. Antibiotic treatment was provided as previously described 20 (link). Briefly, animals were treated with gentamycin (100 mg/L; MPbio), ampicillin (1 g/L; MPbio), erythromycin (10 mg/L; MPbio), vancomycin (0.5 g/L; MPbio), and neomycin (0.5 g/L; MPbio), which were administered via drinking water for 6 weeks. Mice were then divided into six groups for analysis (WT, HZ, WT+CUMS, HZ+CUMS, WT+CUMS+antibiotics, HZ+CUMS+antibiotics).
Gentamycin
Manufactured by MP Biomedicals
Sourced in United States
Gentamycin is a broad-spectrum antibiotic used in laboratory settings. It inhibits bacterial protein synthesis, thereby preventing the growth and reproduction of various bacterial strains.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Erythromycin, by MP Biomedicals (2 mentions)
Ampicillin, by MP Biomedicals (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Human ab serum, by Gemini Bio (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Neomycin, by MP Biomedicals (1 mentions)
Vancomycin, by MP Biomedicals (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
Easysep human na ve cd8 t cell isolation kit, by STEMCELL (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions)
Nalm6, by American Type Culture Collection (1 mentions)
Kanamycin, by MP Biomedicals (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
9 protocols using gentamycin
1
Chronic Unpredictable Mild Stress in Mice
2
Primary T and NK Cell Isolation and Activation
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For T cell preparations, buffy coats were obtained from the New York Blood Center or, alternatively, Leukocyte Reduction System cones (STEMCELL Technologies, 200–0093). T cells were isolated by EasySep™ Human T Cell Isolation Kits (STEMCELL Technologies, 17951). Naïve T cells were isolated by EasySep™ Human Naïve Pan T Cell Isolation Kits (STEMCELL Technologies, 17961). Naïve CD8+ T cells were isolated by EasySep™ Human Naïve CD8+ T Cell Isolation Kit (STEMCELL Technologies, 19258). All isolated T cells were activated by CD3/CD28 Dynabeads (Thermo Fisher, 11132D) at a 1:1 ratio for 2 days. For NK cell preparations, cord blood mononuclear cells were ordered from STEMCELL Technologies (STEMCELL Technologies, 70007.1 or 70007.2). NK cells were isolated by EasySep™ Human NK Cell Isolation Kits (STEMCELL Technologies, 17955) and activated by 100 Gy irradiated K562 Clone 9 feeder cells. Feeder cells were mixed with NK cells at a 2:1 tumor:NK cell ratio. Primary cells were cultured in RPMI 1640 (Gibco, 11875085) with 1% v/v Penstrep (Gibco, 15070–063), 2.5% v/v HEPES (Gibco, 15630–080), 0.02% v/v Gentamycin (MP Biomedicals, 1676245) and 10% v/v heat inactivated human serum AB (GeminiBio, 100–512). Recombinant human IL-2 (PeproTech, 200–02) was added to T cells and NK cells cultures at 50 IU mL−1 or 200 IU mL−1, respectively.
3
Microbial Susceptibility to Gentamycin
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MIC assessment was prepared in 96-well titration micro-plates (Corning Life Science, USA). The wells of the plate were filled with 100 μL of TSB medium. Next, 100 μL of 2000 mg/L of gentamycin (MP Biomedicals, USA) was added to the first of the wells and mixed with the medium. Subsequently, geometric dilution of the antibiotic was performed. Next, 100 μL of bacterial suspension (105 cfu/mL) was introduced to each well. The entire plate was incubated at 37°C/24h in a shaker (Schüttler Microplate Shaker, MTS-4, IKA, Germany). The final range of gentamycin concentrations was 0.98 mg/L–500 mg/L. The culture with no antibiotic added served as a positive control, while the sterility control well contained the sterile medium only. After incubation, 5 μL of triphenyl tetrazolium chloride, TTC (Sigma Aldrich, Germany) was added to each well and incubated for 5 h at 37°C. A change of colorless TTC to red formazan confirmed the presence of metabolically active microorganism. The antibiotic concentration in the first colorless well, neighboring to the red well was taken as the MIC value.
4
Listeria monocytogenes Infection Assay
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L. monocytogenes strain LO28 was thawed and grown overnight a day prior to the experiment in Brain–Heart Infusion (BHI) medium at 37 °C with continuously shaking. After reaching stationary phase, optical density at 600 nm (OD600) was measured and desired multiplicity of infection (MOI) ratio could be determined. An MOI of 100 was used for experiments with RKO cells, while an MOI of 10 was used for experiments performed with COV362 cells. Bacteria were centrifuged 3 min at 10,000g, washed twice with phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium supplemented with 10% (v/v) FBS. Bacteria were added dropwise on top of the target cells. After 1 h of infection, the medium was exchanged to a medium containing a high concentration (50 µg/ml) Gentamycin (MP Biomedicals, Santa Ana, US). After a total of 2 h of L. monocytogenes infection, the medium was changed again, and cells kept in a low concentration of Gentamycin (10 µg/ml).
5
Establishing Cell Lines for CAR-T Research
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293GP cells (Takara Bio, 631458), Nalm6 (ATCC, CRL-3273), and Raji (ATCC, CCL-86) were purchased from commercial vendors. Nalm6-GFP/Luc cells were previously described50 (link) and obtained from the Sadelain lab (MSKCC). Raji-GFP/Luc cells were also previously described85 (link) and obtained from the Brentjen’s lab (MSKCC). K562-CD19 cells were previously described86 (link) and provided under MTA from the Feldman lab (NIH). K562 Clone 9 cells were previously described87 (link) and provided under MTA from the Lee lab (Nationwide Children’s Hospital). Raji and Nalm6 cell lines with stable NLS-mCherry or NLS-GFP reporter expression were developed by retroviral transduction. K562-CD19 FASLG-KO cells were generated by CRISPR gene editing, described below, followed by single cell cloning. All tumor cell lines were cultured in RPMI 1640 (Gibco, 11875085) supplemented with 0.5% v/v Penstrep (Gibco, 15070–063), 1% v/v HEPES (Gibco, 15630–080), 0.02% v/v Gentamycin (MP Biomedicals, 1676245) and 10% v/v heat inactivated fetal bovine serum (GeminiBio, 100–106).
6
Antibiotic Resistance Assessment of Probiotics
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The microdilution protocol was used to assess the resistance of probiotic bacteria to different types of antibiotics [23 (link)] with some modifications according to EFSA Guidance [24 (link)]. The antibiotics ampicillin, chloramphenicol, erythromycin, gentamycin, kanamycin (Sigma Aldrich, St. Louis, MO), and streptomycin (MP Biomedicals, Santa Ana, CA) and the amounts of the active compound were placed into the broth media tube: ampicillin (10 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamycin (10 μg), kanamycin (30 μg), and streptomycin (10 μg). Cultures were inoculated in MRS broth filtered through a 0.22-μm filter and incubating aerobically at 37°C overnight. Furthermore, antibiotic stock solutions were prepared according to the following formula: where P is potency given by the manufacturer (μg /mg), V is volume required (ml), C is final concentration of solution (multiples of 1000, mg/L), and W is weight of antibiotic (mg) to be dissolved in volume V (mL). The data was expressed after 24 hours as the following formula: logN1 is absorbance of culture 620 nm in MRS broth with different antibiotic types and logN0 is absorbance of culture 620 nm in MRS broth as a control.
7
Preparation of Marine BG-11 Media
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Example 2
BG-11 stock solution was purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, Mo.). Stock solutions of the antibiotics spectinomycin (100 mg/mL) and kanamycin (50 mg/mL) were purchased from Teknova (Teknova, Hollister, Calif.). Stock solution of the antibiotic gentamycin (10 mg/mL) was purchased from MP Biomedicals (MP Biomedicals, Solon, Ohio). Marine BG-11 (mBG-11) was prepared by dissolving 35 g Crystal Sea Marinemix (Marine Enterprises International, Inc., MD) in 1 L water and supplementing with BG-11 stock solution. Vitamin B12 (Sigma Aldrich) was supplemented to mBG-11 to achieve a final concentration of 1 μg/L, as needed.
8
HLA-Typed PBMC Isolation and Cell Line Generation
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Leukopaks from HDs were purchased from the New York Blood Center. HLA-typed leukoreduction system chambers from HDs were obtained from the Stanford Blood Center. PBMCs were isolated by density-gradient centrifugation using lymphocyte separation medium (Corning) and cryopreserved until ready for use. High-resolution genomic HLA typing for HD PBMCs was performed by HistoGenetics. The retroviral packaging line 293GP was purchased from Takara Bio (cat. no. 631458), and the HCC70 cell line was purchased from the American Type Culture Collection (cat. no. CRL-2315). Isogenic HCC70 cell lines stably expressing either WT PIK3CA or Mut PIK3CA were obtained by RV infection, followed by subcloning. COS-7 cells were obtained through a material transfer agreement (MTA) from S. A. Rosenberg (National Cancer Institute). Human primary cells were cultured in complete RPMI 1640 (Gibco) media supplemented with antibiotics and 10% human AB serum (GeminiBio). Cell lines were cultured and maintained in RPMI 1640 (Gibco) media supplemented with Pen–Strep (Gibco), gentamycin (MP Biomedicals) and 10% FBS (GeminiBio).
9
Antibiotic Library Preparation
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A total of 12 antibiotics were included in the study as representatives of all major drug classes. Ciprofloxacin (CPR) from MP Biomedicals (Santa Ana, CA) and Gentamycin (GEN), levofloxacin (LVX), tetracycline (TET), tobramycin (TOB), erythromycin (ERY), ampicillin (AMP), clindamycin (CLI), streptomycin (STR), nitrofurantoin (NTR), cefoxitin (FOX), and trimethoprim (TMP)-all from Sigma (St Louis, MO)-were used. Stock solution at 20 mg/mL of each antibiotic was stored in 50μL aliquots at -20°C. Each aliquot was only frozen and thawed once to preserve potency.
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