Antigens printed onto microarrays were glycoproteins or nucleoproteins of HCoVs, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, respiratory syncytial viruses (RSVs), metapneumoviruses (MPVs), parainfluenza viruses (PIVs), adenoviruses (AdVs), and influenza viruses. Protein microarray assays were performed according to the manufacturer’s instructions (Sino Biological, Beijing, China). Briefly, slides were probed with human sera in protein-blocking buffer overnight at 4 °C and labeled with Alexa Fluor® 647 anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) to quantum dot fluorophore for 1 h at room temperature. Dried slides were imaged using the GenePix 4000B microarray scanner (Molecular Devices, San Jose, CA, USA). Mean fluorescence intensity (MFI) of the 4 replicate spots for each antigen was used for the analysis.
Alexa fluor 647 anti human igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor® 647 anti-human IgG is a labeling reagent used for the detection and analysis of human immunoglobulin G (IgG) in various immunological applications. It consists of a highly specific anti-human IgG antibody conjugated to the Alexa Fluor® 647 fluorescent dye. The Alexa Fluor® 647 dye exhibits strong fluorescence and is widely used for flow cytometry, fluorescence microscopy, and other immunoassays.
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Lab products found in correlation
Genepix 4000b microarray scanner, by Molecular Devices (1 mentions)
293fectin reagent, by Thermo Fisher Scientific (1 mentions)
Facs machine, by Beckman Coulter (1 mentions)
Pembrolizumab, by Merck Group (1 mentions)
Nivolumab, by Bristol-Myers Squibb (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 anti mouse igg, by Jackson ImmunoResearch (1 mentions)
V bottom plate, by Corning (1 mentions)
Fortessa, by BD (1 mentions)
3 protocols using alexa fluor 647 anti human igg
1
Protein Microarray Assay for Viral Antibody Detection
2
Characterization of PD-1 Mutants by Flow Cytometry
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Firstly, PD-1 (lacking the intracellular region) was fused with Enhanced green fluorescent protein (EGFP) and then cloned into the pKN009 vector (constructed in our laboratory). The plasmids expressing PD-1 mutants N49A, N58A, N74A, or N116A were created using site-directed mutagenesis. The plasmids were then transfected into HEK 293 cells using 293fectin reagent (Cat.: 12347019, Life Technologies), and the cells were cultured for 24 h, collected, and resuspended in phosphate buffered saline (PBS) at 1 × 107 cells ml−1. Next, the HEK 293 cells expressing wild-type (WT) PD-1 or PD-1 mutants were stained with anti-PD1 MAbs at room temperature for 30 min, washed three times with PBS and then stained with the secondary antibody (Alexa Fluor® 647 anti-human IgG, #109-605-098, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for another 30 min. Following a washing step, cells were analyzed by flow cytometry with a Beckman Coulter FACS machine. Antibodies nivolumab (Lot: AAW4553, Bristol-Myers Squibb) and pembrolizumab (Lot: 6SNL81506, Merck &Co.) were also analyzed in the same way.
3
Fluorescent Labeling of Borrelia burgdorferi
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B. burgdorferi cultures (GFP, wildtype or Δp66) (20 μL/well) were plated in a 96 well v-bottom plate (Corning). After the plate was centrifuged for 10 minutes at 1500 × g at 4°C and the supernatant was aspirated, the B. burgdorferi were resuspended in FACS buffer (30 μL/well) (200 μL; 2% fetal bovine serum in PBS supplemented with EDTA (1 mM, Thermo Fisher Scientific)) containing CV1-G4 or MIAP410 (10 μg/mL). Samples were incubated on ice (30 min) and protected from light. Upon incubation completion, the plate was washed twice with PBS (150 μL). For each wash, bacteria were pelleted prior to aspiration (1500 × g, 10 min, 4°C). After the second wash, the B. burgdorferi were resuspended PBS (30 μL) containing Alexa Fluor 647 anti-human IgG (1:200, Jackson ImmunoResearch) or Alexa Fluor 647 anti-mouse IgG (1:200, Jackson ImmunoResearch). Samples were incubated on ice (30 min) and protected from light. Samples were again washed with PBS (2x) as described above. B. burgdorferi were resuspended in 4% paraformaldehyde (100 μL, EMS) and incubated while protected from light (10 min, 25 °C). Samples were again washed with PBS (2x) as described above prior to resuspension in FACS buffer. Samples were protected from light until analysis on a BD Fortessa.
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