All chemicals were of analytical grade or higher and purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). OD600 measurements were performed by an Eppendorf Biophotometer D30 (Hamburg, Germany). Protein expression was performed in MTP (96-wells, conical bottom) purchased from Greiner Bio-One GmbH (Kremsmünster, Austria) and thereafter incubated in an Eppendorf New Brunswick™ Innova 42 R incubator shaker series (Hamburg, Germany). Pyomelanin absorbance measurements were performed by a VICTOR 3 1420 Multilabel Counter plate reader (Perkin Elmer, Waltham, MA, USA). Plasmid isolation kits were obtained from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). PCRs for SDM were performed with Icycler IQ5 (Bio-Rad). Mastermix reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Victor3 1420 multilabel counter plate reader
Manufactured by PerkinElmer
Sourced in United States
The VICTOR3 1420 multilabel-counter plate reader is a versatile laboratory instrument designed for high-throughput analysis. It is capable of performing various types of assays, including absorbance, fluorescence, and luminescence, across a range of microplate formats. The VICTOR3 1420 provides reliable and accurate data for a wide array of applications in life science research and drug discovery.
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Lab products found in correlation
Horseradish peroxidase conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Biophotometer d30, by Eppendorf (1 mentions)
Icycler iq5, by Bio-Rad (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
D4540, by Merck Group (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Ldh release assay kit, by Promega (1 mentions)
Vibrio cholera neuraminidase vcna, by Roche (1 mentions)
8 protocols using victor3 1420 multilabel counter plate reader
1
Protein Expression and Pyomelanin Assay
2
Influenza Virus Receptor Affinity Assay
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Receptor affinity was determined in a solid-phase direct virus-binding assay as previously described. In brief, influenza viruses were bound to fetuin-coated plates at 4 ˚C overnight. Biotinylated glycans (a2,3’SL; a2,6’SL; or a2,6’SLN; Glycotech Corporation, Gaithersburg, MD, USA) were added to influenza-coated plates at varying dilutions and incubated for 4 h. Glycan binding was detected by adding horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) followed by 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich, St Louis, MO, USA); the resulting absorbance at 450 nm was measured in a VICTOR3 1420 multilabel-counter plate reader (Perkin-Elmer, Waltham, MA, USA).
3
Influenza Virus Receptor Binding Assay
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The receptor-binding preference of the AI virus isolates was determined using a solid-phase direct virus-binding assay as previously described [16 ]. Briefly, wild bird AI viruses were bound to fetuin-coated 96-well microplates at 4°C overnight. Polyacrylamide (PAA)-biotin-conjugated glycans Neu5Acα2–3Galβ1–4Glc β1 (α2,3’-SL-PAA-biotin) or Neu5Acα2-6Galβ1-4GlcNAc (α2,6’SLN-PAA-biotin) (Glycotech Corporation, Gaithersburg, MD, USA) were added to influenza-coated plates at varying dilutions and incubated for an additional 4 h. Glycan binding was detected by adding horseradish peroxidase (HRP)-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA), and absorbance at 450 nm was measured via a VICTOR3 1420 multilabel-counter plate reader (Perkin-Elmer, MA, USA). The receptor-binding specificity of the 2009 pandemic H1N1 virus was also determined and compared as a positive control for binding preference to mammalian virus receptors.
4
Virus-Glycan Binding Assay Protocol
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Receptor affinity was determined by using a solid-phase direct virus-binding assay, as previously described (17 (link)). In brief, influenza viruses were bound to fetuin-coated plates at 4°C overnight. Biotinylated glycans (α2,3′-sialylated glycans. α2,6′-sialylated glycans, or α2,6′-sialyl lactosamine; Glycotech Corporation, Gaithersburg, MD) were added to influenza virus-coated plates at various dilutions and incubated for an additional 4 h. Glycan binding was detected by adding horseradish peroxidase (HRP)-conjugated streptavidin (Invitrogen, Carlsbad, CA), followed by 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma, St. Louis, MO), and the absorbance at 450 nm was measured by using a Victor3 1420 multilabel counter plate reader (PerkinElmer, MA, USA).
5
MTT Assay for Cell Viability
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The (3‐4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide solution at 1 mg/mL (MTT; M2128‐5G, Sigma‐Aldrich, France) was added to each well after preliminary collection of culture supernatants. The plates were incubated for 3 hours at 37°C, 5% CO2, and saturated humidity. The MTT solution was carefully removed from the wells, and 100 µL of dimethyl sulfoxide (DMSO; D4540, Sigma‐Aldrich) was added to each well. The plates were gently swirled for 5 minutes. The optical density was read at 540 nm with a Victor3™ 1420 Multilabel Counter Plate Reader (PerkinElmer) and Wallac Workstation software. The cellular viability of each well was normalized relative to the corresponding control‐supplemented KBM (without hydrocortisone) in an inflammatory condition (100% viability). Only data obtained with a cell viability threshold of above 80% were analyzed.
6
Bacterial Growth Inhibition Assay
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At least four replicates of each sample and control were run on PerkinElmer Victor 31420 Multilabel Counter Plate Reader. As a background and positive control, pure LB media and the bacteria growing in LB media only were used. The tests were premixed in a 2 ml microcentrifuge tube, containing a total of 2 ml of solution, including 30 μl of bacteria inoculum and precalculated volumes of nanoparticle samples as well as Syn71 peptide alone. In addition, background solutions for each nanoparticle sample and concentration without bacteria were prepared. The solutions were mixed thoroughly, and 200 μl of the solution was pipetted into each well of a sterile 96-well plate and incubated under continuous shaking at 37 °C for 24 h with OD600 nm measurements taken every 10 min. The resulting growth curves were corrected with the LB media background as well as the nanoparticle backgrounds.
7
Cytotoxicity Assay for Lung NK and CD8+ T Cells
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To isolate the CD8+ T cells and NK cells, total lung cells were first digested in collagenase digestion solution. Both cell types were magnetically separated via negative selection with a CD8+ T-cell isolation kit (Miltenyi Biotec; CA, USA) and positive selection with a CD49b (DX5) kit (Miltenyi Biotec). The cell-mediated cytotoxicity assay was performed using the EL-4 cells as target cells [44] and quantified by using the lactate dehydrogenase (LDH)-release assay kit according to the instructions (Promega; CA, USA). In brief, lung NK cells and CD8+ T cells were mixed with target cells at an effector cell to target cell ratio of 50:1 and incubated at 37 °C for 4 h. To determine the CD8+ T cells’ cytotoxicity, target cells were stimulated with the 50 % tissue culture-infective dose (TCID50) of UV-inactivated virus for 24 h. Each sample was incubated with 50 μl of substrate at room temperature for 30 min, and optical density (OD) values were measured at 490 nm wavelength by using the VICTOR3 1420 multilabel counter plate reader (Perkin-Elmer; MA, USA). The cytotoxicity percentage was calculated by using the following formula: (mean experimental OD value–mean spontaneous OD value)/(mean maximal OD value–mean spontaneous OD value) × 100.
8
Influenza Virus Receptor Binding Assay
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Receptor affinity was determined in a solid-phase direct virus-binding assay as previously described.22 (link) In brief, influenza viruses were bound to fetuin-coated plates at 4 °C overnight. Biotinylated glycans (α2,3′SL; α2,6′SL; or α2,6′SLN; Glycotech Corporation, Gaithersburg, MD, USA) were added to influenza-coated plates at varying dilutions and incubated for 4 h. Glycan binding was detected by adding horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) followed by 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich, St Louis, MO, USA); the resulting absorbance at 450 nm was measured in a VICTOR3 1420 multilabel-counter plate reader (Perkin-Elmer, Waltham, MA, USA). The receptor binding specificities of HPAI H5 viruses were also determined in HA assays using 0.5% re-sialylated chicken red blood cells (cRBCs). For the HA assay, sialic acid residues were enzymatically removed from cRBCs by incubation of the cells with 50 mU Vibrio cholera neuraminidase (VCNA: Roche, San Francisco, CA, USA) at 37 °C for 1 h, the cells were then re-sialyated by incubation with either 2,6-(N)-sialyltransferase or 2,3-(N)-sialyltransferase (Sigma-Aldrich, St Louis, MO, USA) at 37 °C for 4 h.23 (link)
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