The production of ROS by E. faecalis ATCC 700802 after treatment with pyochelin was evaluated using a peroxynitrite indicator, 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, UK), which can detect a broad range of ROS including nitric oxide and hydrogen peroxide [14] . The adjusted bacterial culture (0.5 McFarland exponential phase bacteria culture) were treated with different concentrations of pyochelin corresponding to 1, 2 and 4 times MIC in presence of DCFH-DA at a final concentration of 5 µM in 0.85% saline and incubated at 37 °C aerobically at 200 rpm (Smith, A3555, Progressive Scientific) for 24 h. Untreated bacterial culture was served as a negative control. The fluorescence emission of DCFH-DA was measured at 525 nm using a Tecan microtitre plate reader with an excitation wavelength of 485 nm [15] . The background fluorescence of 0.85% saline and autoflorescence of the bacterial cells incubated without the probe was measured to calculate the net fluorescence emitted from the assay itself. Experiment was conducted in triplicate.
Microtitre plate reader
Manufactured by Tecan
Sourced in Austria
The Microtitre plate reader is a laboratory instrument used to measure and analyze the optical properties of samples in a microtitre plate. It is capable of detecting and quantifying various types of signals, including absorbance, fluorescence, and luminescence, from samples in the microplate wells.
Automatically generated - may contain errors
Lab products found in correlation
Human prothrombin, by Enzyme Research (3 mentions)
Dcfh da, by Merck Group (1 mentions)
Dcfh da, by Tecan (1 mentions)
Ab42632, by Abcam (1 mentions)
Ab101661, by Abcam (1 mentions)
Cpass neutralization antibody detection kit, by GenScript (1 mentions)
Para nitrophenyl phosphate, by Merck Group (1 mentions)
5 protocols using microtitre plate reader
1
Pyochelin-Induced ROS Production in E. faecalis
2
HDAC Enzyme Activity Assay
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The HDAC (1, 6, 7) enzymes were obtained from Abcam (ab101661 for HDAC1, ab42632 for HDAC6, and ab101660 for HDAC7). The HDAC3 enzyme (BML-SE515-0050) and HDAC8 enzyme (H90-30H-05) were bought from ENZO Inc. (New York, NY) and SignalChem (Richmond, Canada), respectively. First, the reaction mixture consisted of 25 mM Tris, 1 mM MgCl2, 2.7 mM KCl, 137 mM NaCl, 0.1 mg/mL BSA, and HDAC enzymes (7.2 ng/well, 3.4 ng/well, 15.0 ng/well, and 22.0 ng/well of HDAC1, HDAC3, HDAC6, and HDAC8). The test compounds were diluted by DMSO. Second, the diluent and HDAC enzymes were added to the plate and incubated for 15 min. Then, the substrates of HDAC enzymes (Ac-Leu-Gly-Lys (Ac)-AMC for HDAC1/3/6; Ac-Leu-Gly-Lys (Tfa)-AMC for HDAC8) were added and incubated for half hour in room temperature. Finally, HDAC assay developer was used to quench the reaction for half hour. The fluorescence of the reaction was analysed using a TECAN microtitre plate reader (λEx: 350–360 nm, λEm: 450–460 nm).
3
Immunoglobulin Serum Quantification
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Absolute serum concentrations of the different immunoglobulins were measured using Human IgG, IgM and IgA Enzyme‐linked Immunosorbent Assay (ELISA) Kits (Invitrogen, Cat. No. BMS2091, BMS2098, BMS2096) as per the manufacturer’s instructions. Absorbance was read at 450 nm with a reference wavelength of 620 nm in a microtitre plate reader (Tecan, Morrisville, NC).
4
Neutralizing Antibody Detection Against SARS-CoV-2
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Neutralising activity of patient sera was assessed using the cPass Neutralization Antibody Detection Kit (GenScript, Cat. No. L00847‐A), which is a surrogate test detecting circulating neutralising antibodies against SARS‐CoV‐2 that block the interaction between the receptor‐binding domain (RBD) of the viral spike glycoprotein with the ACE2 cell surface receptor. Briefly, samples and controls were diluted with sample dilution buffer and pre‐incubated with the Horseradish peroxidase (HRP) conjugated recombinant SARS‐CoV‐2 RBD fragment (HRP‐RBD) or one of its variants listed in Supplementary table 2 to allow the binding of the circulating neutralisation antibodies to HRP‐RBD. The mixture was then added to the capture plate, which was pre‐coated with the hACE2 protein. The unbound HRP‐RBD as well as any HRP‐RBD bound to non‐neutralising antibody was captured on the plate, while the circulating neutralisation antibodies HRP‐RBD complexes remained in the supernatant and were removed during washing. Following a wash cycle, TMB substrate solution was added followed by the Stop Solution. The absorbance of the final solution was read at 450 nm in a microtitre plate reader (Tecan).
5
Antiphospholipid Antibody Measurement
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This was performed following previously described protocol and validated method [11 (link)]. Specifically, the assays average inter- and intra-assay coefficients of variations were <3.3% and <8.2%, respectively. The diagnostic specificity for APS was 92.5% and the diagnostic sensitivity was 59.0%. The diagnostically relevant cut-off of aPS/PT was set on the 99th percentile of 222 blood donors. Briefly, phosphatidylserine was coated on polystyrene microtitre plates (medium binding, Costar, Cambridge, MA, USA) and incubated overnight at 4°C. After blocking with Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 5 mM CaCl2 (1% BSA-TBS-Ca++) plates were washed in TBS containing 0.05% Tween-20. Human prothrombin (10 mg/L) (Enzyme Research Laboratories, UK) and patients' sera diluted 1 : 100 in 1% BSA-TBS-Ca++ were applied to wells immediately one after the other and incubated for 1 hour at room temperature (RT). Afterwards, plates were washed and incubated with alkaline phosphatase-conjugated goat anti-human IgG or IgM (ACSC, Westbury, USA) for 30 minutes at RT. After the last wash para-nitrophenylphosphate (Sigma Chemical Company, USA) in diethanolamine buffer (pH 9.8) was applied as substrate and OD405 was kinetically measured by microtitre plate reader (Tecan, Grödig, Austria).
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This was performed following previously described protocol and validated method [11 (link)]. Specifically, the assays average inter- and intra-assay coefficients of variations were <3.3% and <8.2%, respectively. The diagnostic specificity for APS was 92.5% and the diagnostic sensitivity was 59.0%. The diagnostically relevant cut-off of aPS/PT was set on the 99th percentile of 222 blood donors. Briefly, phosphatidylserine was coated on polystyrene microtitre plates (medium binding, Costar, Cambridge, MA, USA) and incubated overnight at 4°C. After blocking with Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 5 mM CaCl2 (1% BSA-TBS-Ca++) plates were washed in TBS containing 0.05% Tween-20. Human prothrombin (10 mg/L) (Enzyme Research Laboratories, UK) and patients' sera diluted 1 : 100 in 1% BSA-TBS-Ca++ were applied to wells immediately one after the other and incubated for 1 hour at room temperature (RT). Afterwards, plates were washed and incubated with alkaline phosphatase-conjugated goat anti-human IgG or IgM (ACSC, Westbury, USA) for 30 minutes at RT. After the last wash para-nitrophenylphosphate (Sigma Chemical Company, USA) in diethanolamine buffer (pH 9.8) was applied as substrate and OD405 was kinetically measured by microtitre plate reader (Tecan, Grödig, Austria).
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