The hemagglutination assay was used to assess functionality of rHA to agglutinate erythrocytes. Hemagglutination assays were performed similarly to previously described protocol [24 (link)]. In brief, 50 μL of duplicate two-fold serial dilutions of representative wild type rHA from A/Singapore/6/86 (Sing/86) and A/New Caledonia/20/99 (NC/99), their corresponding Y98F mutant versions (HAΔSA) and influenza viruses (positive controls), and BSA (negative control) were diluted in PBS, starting from 50 μg/mL for proteins or 5.25 × 108 and 4.1 × 108 plaque forming units per milliliter (PFU/mL) for Sing/86 and NC/99 influenza viruses, respectively, and incubated for 30 min with an equal volume of 0.8% turkey erythrocytes diluted in PBS. Erythrocytes were washed and used the day of the assay. The plates were mixed by agitation and covered, and the erythrocytes were settled for 30 min at RT. The hemagglutination titer was determined by the reciprocal dilution corresponding to the rHA concentration of the last well that contained agglutinated erythrocytes. Images of hemagglutination assays were acquired using the ImmunoSpot S6 ULTIMATE (Cellular Technology Limited, Shaker Heights, OH, USA).
Immunospot s6 ultimate
Manufactured by Cellular Technology
Sourced in United States
The ImmunoSpot S6 ULTIMATE is a laboratory instrument designed for the detection and enumeration of specific immune cells, such as T cells and B cells, using the enzyme-linked immunospot (ELISpot) assay. The device provides precise and reliable measurements of the frequency of antigen-specific cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Multiscreen high protein binding immobilon p membrane plates, by Merck Group (1 mentions)
Complete rpmi 1640, by Southern Biotech (1 mentions)
Alp goat anti mouse igg, by Southern Biotech (1 mentions)
Immunospot software, by Cellular Technology (1 mentions)
5 bromo 4 chloro 3 indolylphosphate, by Bio-Rad (1 mentions)
3 protocols using immunospot s6 ultimate
1
Hemagglutination Assay for Influenza rHA
2
Quantifying Tetanus Toxoid-Specific Antibody-Secreting Cells
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TT-specific ASC were enumerated by ELISPOT, as previously described (15 (link), 19 (link), 20 (link), 38 (link)). MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 10 μg/ml TT overnight at 37°C, blocked with complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.). Duplicates of cells from spleen and bone marrow in four three-fold dilutions starting with 1 × 107 cells in 100 μL in complete RPMI 1640 per well were incubated for 5 hours at 37°C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4°C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). The number of spots, each representing a cell secreting TT-specific IgG antibodies, was counted with ELISPOT reader ImmunoSpot® S6 ULTIMATE using ImmunoSpot® SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).
3
Gsα Peptide-Specific T Cell IFN-γ ELISPOT
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If cell numbers were sufficient with either freshly peptide prestimulated or thawed prestimulated T cells, IFN-y ELISPOT assays (CTL Europe GmbH, Bonn, Germany) were performed. T cells and autologous PBMCs were plated onto plates precoated with antihuman IFN-y at 1:1 ratio (0.5–1.0×105 cells) and 10 µM of 30 mer Gsα peptides (WT, R201C, and R201H) were added. Wells with no stimulus served as a negative control, while cells stimulated with 0.1 µg/mL SEC3 served as a positive control. Plates were incubated for 24 hours prior to addition of detection reagents and substrate following the manufacturer’s instructions. The plates were imaged and the spot counts determined using an automated ELISPOT analyzer ImmunoSpot S6ULTIMATE (Cellular Technology, Shaker Heights, USA). Peptide-specific spot counts were determined by subtracting the mean spot number of no peptide-stimulated coincubated T cells and PBMC control wells for each patient from the number of spots in the Gsα peptide (WT, R201C, and R201H) stimulated wells. As there were insufficient cell numbers to perform titrations (except for one patient), saturated well counts for SEC3-positive controls where spots were too numerous to count (TNT) were set to >1000 spots or TNTC. Raw data included images for each well for visual quality checking (online supplemental figure 1 ).
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