LNs (bilateral inguinal) and bone marrow cells from both tibia were harvested from vaccinated BALB/c mice (n = 4 per group) and single cell suspensions were prepared. The cells were used in an IL-4 ELISPOT assay (Mouse IL-4 ELISpotPLUS (3311-4APW, Mabtech, Nacka Strand, Sweden), performed according to kit instructions. Cells were stimulated with HA(PR8) MHC class I (IYSTVASSL) or class II (SVSSFERFEIFPK or HNTNGVTAACSHEG)-restricted peptides (Proimmune, Oxford, England), or full-length HA [A/Puerto Rico/8/34 (H1N1)] (11684-V08H, Sino Biological). For the B-cell ELISPOT, MultiScreen HTS filter plates (MSIPS45, Merck Millipore Ltd., Tullagreen, Ireland) were coated with 0.5 µg/well of recombinant HA (PR8) (11684-V08H, Sino Biological) overnight at 4 ℃. Bone marrow suspensions were seeded and incubated for 20 h. Spots were detected with anti-mouse IgG (A1418, Sigma-Aldrich) 1:5000 and developed with phosphatase substrate (P4744, Sigma-Aldrich). ELISPOT plates were analyzed in CTL-ImmunoSpot® analyzer (CTL, Shaker Heights, OH, USA).
P4744
Manufactured by Merck Group
Sourced in United States
P4744 is a laboratory instrument produced by Merck Group. It is designed for conducting various scientific experiments and analyses in a controlled laboratory environment. The core function of this equipment is to facilitate the measurement and analysis of samples, without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Multiscreen hts filter plates, by Merck Group (2 mentions)
Anti mouse igg a1418, by Merck Group (2 mentions)
A3687, by Merck Group (2 mentions)
A3562, by Merck Group (2 mentions)
Biotek epoch 2, by Agilent Technologies (2 mentions)
Envision 2104 multilabel reader, by PerkinElmer (1 mentions)
Infinite 200 pro nanoquant, by Tecan (1 mentions)
Elisa plate, by Corning (1 mentions)
Synergy h4 hybrid, by Agilent Technologies (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
A9316, by Merck Group (1 mentions)
Spark plate reader, by Tecan (1 mentions)
12 protocols using p4744
1
BALB/c Mice Vaccine Response
2
Measuring Kinase and Phosphatase Activities
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For measuring the in vitro kinase activity of CDK5, purified proteins were incubated with active CDK5/p35 complex (Merck Millipore) in kinase reaction buffer (50 mM Tris at pH 7.5, 5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, and [γ-32P]ATP for 30 min at 30 °C. For measuring the in vitro phosphatase activity of VHR, purified proteins were further incubated with 5 × 30 mM of the phosphatase substrate p-nitrophenol phosphatase hexahydrate (P4744, Sigma-Aldrich) dissolved in recommended buffer (1 M diethanolamine at pH 10.4 with 0.5 mM MgCl2) for 2 h at 37 °C or with recombinant p-ERK2 (gift from H.S. Yoon, Nanyang Technological University, Singapore) for 1 h at 37 °C.
3
ELISA Protocol for Influenza Antibody Detection
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High binding 96 well plates (3590, Corning) were coated with 1 µg/ml anti-A/B mAb, or anti-IAV H1N1 (A/Puerto Rico/8/1934) HA (clone H36-4-52, produced from hybridoma provided by Siegfried Weiss62 (link) mAb in PBS, incubated at 4 °C overnight, and blocked with 200 µl blocking buffer (1% w/v BSA in PBS with 0.02% w/v Na-Azide) per well. Supernatants from transfected HEK293E cells were added (50 µl/well) and serially diluted 3-fold in triplicates with ELISA buffer (0.1% w/v BSA and 0.2% Tween 20 in PBS/Na-Azide). Plates were incubated for 2 hours at room temperature. Vaccine proteins were detected for 1 hour with 1 µg/ml biotinylated anti-H1 mAb, a cross-reactive rabbit mAb specific for IAV HA stem (1:3000, clone #2, 86001-RM01, Sino Biological), or 1 µg/ml rabbit-anti-AIV H3N2 (A/Brisbane/10/2007) HA mAb (clone# 104, 11056-R104, Sino Biological). Biotinylated mAb was detected with streptavidin-Alkaline Phosphatase (ALP) conjugate (1:3000, RPN1234, Southern Biotech) and rabbit mAbs with polyclonal (p) anti-rabbit IgG Ab-ALP conjugate (1:3000, A3687, Sigma Aldrich). Signals were developed with phosphatase substrate (1 mg/ml, P4744, Sigma) dissolved in diethanolamine substrate buffer. OD405nm was measured using EnVision 2104 Multilabel Reader with EnVision Manager 1.12 software (PerkinElmer).
4
Measuring Phosphatase Activity of VHR
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For measuring the in vitro phosphatase activity of VHR using the phosphatase substrate p-nitrophenyl phosphate (pNPP), 5 μg of GST-VHR and 10 μg of His-HSP70 were incubated in reaction buffer (50 mM Tris at pH 7.5, 5 mM MgCl2, 0.5 mM DTT, and 150 mM KCl) for 30 min at 30 °C. Purified proteins were further incubated with 5 × 30 mM of pNPP (P4744, Sigma-Aldrich) dissolved in recommended buffer (1 M diethanolamine at pH 10.4 with 0.5 mM MgCl2) for 2 h at 37 °C. The hydrolysis of the substrate was measured at 405 nm using an ELISA reader with Infinite 200 Pro NanoQuant (TECAN). For measuring the in vitro phosphatase activity of VHR using recombinant p-ERK2, 1 μg of GST-VHR and 2 μg of His-HSP70 were incubated in reaction buffer (50 mM Tris at pH 7.5, 5 mM MgCl2, 0.5 mM DTT, and 150 mM KCl) for 30 min at 30 °C. Purified proteins were further incubated with 50 ng recombinant p-ERK2 (Merck Millipore) for 1 h at 37 °C.
5
Serological Profiling of H7N9 Influenza
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Blood was harvested by puncture of the saphenous vein, and sera were isolated by centrifugation. ELISA plates (Costar 3590) were coated with 0.5 μg/ml recombinant HA [A/Shanghai/1/2013 (H7N9)] (40104-V08B; Sino Biological), blocked with 1% BSA, and incubated with serially diluted serum samples assayed individually (n = 6 to 12/group). HA-specific antibodies were detected with alkaline phosphatase conjugated goat anti-mouse IgG (A1418; Sigma-Aldrich), developed with phosphatase substrate (P4744; Sigma-Aldrich) and analyzed as previously described. For all serum ELISAs, titers were determined as the last serum dilution with an optical density at 405 nm above background (the mean absorbance from NaCl-vaccinated mice added five times the standard error of the mean for the group).
6
Enzyme-Linked Immunosorbent Assay for Protein Detection
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Isolated proteins from the tissue samples were coated overnight at 4 °C onto NUNC Maxisorp enzyme-linked immunosorbent assay (ELISA) plates at 2 µg/mL and 100 µL/well. The coating buffer was 0.1 M NaHCO3, pH 9. The following day, the wells were washed with PBS supplemented with 0.01% Tween, and the residual binding sites were blocked with 5% PBS-milk. The wells were washed again, and 100 µL primary antibodies (1:2000; same as those used during the immunoblotting) were put into each well. After 1 h incubation at room temperature, the wells were washed again and secondary anti-mouse (A3562, Sigma Aldrich) and anti-rabbit (A3687, Sigma Aldrich) IgG whole molecule alkaline phosphatase antibodies produced in goat were prepared at 1:2000 dilution in 1% PBS-milk, and were applied (100 µL/well). After 1 h incubation at room temperature, the wells were washed again, and 100 µL/well alkaline phosphatase substrate (P4744; Sigma Aldrich) was added. Signals were measured using a microplate reader (Synergy H4 Hybrid; BioTek, Winooski, VT, USA) at 405 nm.
7
Alkaline Phosphatase Activity Assay
8
Influenza Antibody Avidity Assay
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Blood was harvested by puncture of the saphenous vein, and sera collected by centrifugation. ELISA plates (Costar 5390, Corning, Corning, NY) were coated with 0.5µg/ml rec. HA from A/California/07/2009 (11085-V08H, Sino Biological, Inc., Wayne, PA, USA), blocked with 2% BSA/PBS, and incubated with serially diluted serum samples assayed for individual mice. Captured serum antibodies were detected with anti-mouse IgG1-bio (553500, BD Pharmingen, San Diego, CA, USA), or anti-mouse IgG2a- bio (553502, BD Pharmingen), and streptavidin-alkaline phosphatase (RPN1234, GE Healthcare, Buckinghamshire, UK), or alkaline phosphatase conjugated goat anti-mouse IgG (A2429, Saint-Louis, MO, USA). Plates were developed with phosphatase substrate (P4744, Sigma-Aldrich).
Resistance to UREA wash was used to calculate avidity index. Captured serum antibodies were incubated for 10min with 2M UREA or PBS before detecting remaining serum antibodies with alkaline phosphatase conjugated goat anti-mouse IgG (A9316, Sigma Aldrich). AUC was calculated for the dilution curves and baseline for AUC was calculated based on NaCl serum levels. Avidity index is defined as AUC for samples treated with UREA divided by AUC for the corresponding PBS treated sample.
Resistance to UREA wash was used to calculate avidity index. Captured serum antibodies were incubated for 10min with 2M UREA or PBS before detecting remaining serum antibodies with alkaline phosphatase conjugated goat anti-mouse IgG (A9316, Sigma Aldrich). AUC was calculated for the dilution curves and baseline for AUC was calculated based on NaCl serum levels. Avidity index is defined as AUC for samples treated with UREA divided by AUC for the corresponding PBS treated sample.
9
Bone Marrow Cell Recall Assay
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Bone marrow was harvested from femur and tibia. Single cell suspensions were prepared and seeded on MultiScreen HTS filter plates (MSIPS45, Merck Millipore Ltd., Tullagreen, Ireland) pre-coated overnight at 4°C with 0.5 μg/well of rec.HA (Cal07) (11085-V08H, Sino Biological), and incubated for 20h. Spots were detected with anti-mouse IgG (A1418, Sigma-Aldrich), developed with phosphatase substrate (P4744, Sigma-Aldrich) and analyzed in CTL- ImmunoSpot® analyzer (CTL, Shaker Heights, OH, USA).
10
Quantifying Biotinylated Proteins by ELISA
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Both as a check on Bradford-based molarity calculations and for determining how to form cell-staining tetramers, biotinylated recombinant proteins were quantified by a competitive ELISA. Dilution series of the target proteins were made in 96-well plates, then incubated with a known constant quantity of a conjugated avidin (SAV–HRP for binding assay calculations, SAV–PE for cell-binding cytometry reagents and neutravidin for immune cell perturbation assays). After at least 45 min at 20 °C, these pre-incubated samples were transferred to a BSA-blocked streptavidin-coated 96-well plate (Greiner 655990). After another 45 min or longer incubation, plates were washed three times with HBS and 0.1% (v/v) Tween-20. Primary antibody, typically 1 µg ml−1 of an OX68 monoclonal against the protein epitope tags51 (link), was prepared in 100 µl 2% (m/v) BSA in HBS, incubated for 60 min and then washed three times. Secondary antibody of goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma A3562) was similarly incubated at 0.2 μg ml−1 for 30 min before another three washes were performed. Finally, 60 µl of 1.5 mg ml−1p-nitrophenyl phosphate (Sigma P4744) in diethanolamine buffer was added and the absorbance at 405 nm was measured after 30–60 min by a Tecan Spark plate reader.
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