Assay was performed as previously described (Cebrian et al., 2011 (link)), with some modifications using the LiveBLAzer FRET-B/G Loading Kit with CCF4-AM (Thermo Fisher Scientific, K1095). LPS-treated BM-derived DCs were loaded with 1 μM of CCF4-AM at room temperature, followed by incubation with 2 mg/mL β-lactamase (Sigma, P0389) for the indicated time. Cells were subsequently analyzed by flow cytometry, and blue-to-green (excitation 405 nm, emission 450 nm/525 nm). FRET ratio was used as an indicator for efficiency of antigen export into the cytosol. Response ratios were calculated as per manufacturer’s instructions and normalized to the signal intensity of control cells, which had been incubated on ice.
β lactamase
Manufactured by Merck Group
Sourced in United Kingdom
β-lactamase is an enzyme that hydrolyzes the β-lactam ring of certain antibiotics, such as penicillins and cephalosporins, rendering them ineffective. It is a widely studied enzyme in the field of microbiology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
β lactamase, by Thermo Fisher Scientific (2 mentions)
Liveblazer fret b g loading kit with ccf4 am, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Nitrocefin, by Merck Group (1 mentions)
Buffered peptone water broth, by Thermo Fisher Scientific (1 mentions)
β galactosidase, by Merck Group (1 mentions)
Rapid alkaline phosphatase, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar, by Hardy Diagnostics (1 mentions)
Clavulanic acid, by Merck Group (1 mentions)
Mueller hinton agar (mha), by Merck Group (1 mentions)
Maxsignal β lactam elisa kit, by PSC Biotech (1 mentions)
Tryptic soy broth (tsb), by Hardy Diagnostics (1 mentions)
Ethanol, by Merck Group (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
6 protocols using β lactamase
1
Cytosolic Antigen Delivery Quantification
2
Measuring Cellular β-Lactamase Activity
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The CCF4/β-lactamase assay was performed in Willco petri dishes according to the protocol of the manufacturer (K1095, Life Technologies) and as described previously (Dingjan et al., 2016 (link)). Briefly, DCs were incubated with 1 μM Substrate Loading Solution and 1 μg/ml LPS as adjuvant for 90 min, then washed and incubated with 1 mg/ml β-lactamase (from Pseudomonas aeruginosa, expressed in E. coli; L6170, Sigma) and 1 μg/ml LPS for 3 h. Cells were washed again and imaged with a Leica SP8 confocal microscope equipped with a 63 × 1.20 NA water immersion objective (405 nm excitation; 419–458 nm blue and 504–600 nm green emission of the coumarin donor and fluorescein acceptor fluorophores, respectively). The FRET cleavage was calculated as the ratio of the green over the blue fluorescence intensities. The FRET efficiency of cells in absence of β-lactamase was used as ‘no cleavage’ control.
3
Antigen Export Quantification by FRET-Based Assay
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Measurement of antigen export to the cytosol was performed as previously described32 (link). Briefly, cells were loaded with 4 μM CCF4-probe (a FRET-sensitive cytosolic substrate of β-lactamase purchased from Life Technologies) for 1 h, were subsequently washed with PBS and incubated at 37 °C with 2 mg/mL β-lactamase (Sigma) for different time points. Reactions were stopped with cold PBS and live, single, CD11c+MHCII+ cells were analyzed by flow cytometry by monitoring the increase in 450 nm fluorescence resulting from CCF4 cleavage and loss of 535 nm emission fluorescence.
4
Antimicrobial Activity Evaluation
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Bacterial strains of E. coli (NCTC 12241, 11560, 13351) and S. aureus (NCTC 12981, 12973) were obtained from Pro-Lab Diagnostics (UK). Nitrocefin was obtained from Merck Millipore (UK). Chlorophenol red-β-D-galactopyranoside (CPRG), 5-bromo-6-chloro-3-indolyl phosphate p-toluidine salt (BCIP, magenta phosphate) or magenta phosphate, β-galactosidase, β-lactamase and rAPid alkaline phosphatase were all acquired from Sigma-Aldrich (UK). HMRZ-86 was purchased from Bio-Rad Laboratories (UK). Filter paper (Whatman grade 4) was bought from GE Healthcare Life Sciences (UK). Mueller-Hinton broth and Buffered Peptone Water broth were purchased from Oxoid Ltd, UK. Pasteurised cow's milk was purchased from local stores (Hull, UK).
5
Antibiotic Susceptibility Testing
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Tryptic soy broth and tryptic soy agar were obtained from Hardy Diagnostics (Santa Monica, CA). Antibiotics, Mueller Hinton agar, clavulanic acid, β-lactamase (from Enterobacter cloacae), and ethanol were obtained from Sigma-Aldrich Chemicals. The β-lactam ELISA kit (MaxSignal® β-Lactam ELISA Kit) was obtained from Bioo Scientific.
6
Quantifying Intracellular Lactamase Activity
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Control or ESCRT-III-deficient MutuDC were seeded at 0.2 × 106 cells/well in flat-bottom 96-well plates and incubated for 20min or 2h with or without 2 mg/mL β-lactamase (#P0389, Sigma-Aldrich), at 37°C. Cells were then washed three times with ice-cold PBS, detached and resuspended in cold buffer containing 50 mM NaH2PO4, 300mM NaCl, 10mM imidazole and 5mM mercaptoethanol pH = 8 (Cebrian et al., 2011 (link)) at a concentration of 1 × 106 cells/100 μL buffer. Samples were then sonicated (5 rounds of sonication, 30s ON, 30s OFF each) on Bioruptor Pico (Diagenode) and centrifuged at 16 000g, 15 min, 4°C to remove insoluble material. Lysates were then incubated for 1h, at 37°C, with 100 μM nitrocefin (#484400-5MG, Calbiochem), a chromogenic lactamase substrate that undergoes distinctive color change from yellow to red (486 nm) upon lactamase hydrolysis. After 1h of incubation, optical density at 490 nm was measured by spectrophotometry. Soluble β-lactamase at 5 mg/mL was used as a positive saturation control.
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