The following antibodies were used for flow cytometric staining: anti-IAV NP (Abcam, ab20921), Mouse IgG1 isotype control (Abcam, ab91356). Intracellular staining was performed using the BD Cytofix/Cytoperm kit (Becton Dickinson, 554714), according to the manufacturers instructions. All samples were run on a Guava easyCyte HT cytometer (Millipore) and analyzed using Flowjo (version 10.4.2, TreeStar)
Ab20921
Manufactured by Abcam
Sourced in United Kingdom
Ab20921 is a recombinant monoclonal antibody product developed by Abcam. It is designed for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte ht cytometer, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Ficoll paque plus density gradient, by GE Healthcare (1 mentions)
Sialidase, by Merck Group (1 mentions)
Control igg, by GeneTex (1 mentions)
Dmi3000b microscope, by Leica (1 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
5 protocols using ab20921
1
Flow Cytometric Analysis of IAV NP
2
Antibody-Dependent Cell-Mediated Phagocytosis of Influenza Virus
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Isolation of human neutrophils from peripheral blood was carried out by using Ficoll-Paque PLUS density gradient (GE Healthcare) according to the procedures described previously [51 (link)]. ADCP by THP1 cells (from ATCC) and neutrophils was performed essentially as a previous report [52 (link)]. Control IgG was purchased from GeneTex (GTX16193). Briefly, Cal/09 virus was incubated with indicated antibodies at 37°C for 1 h and added to the sialidase (0.5 unit/mL, Sigma) pre-treated THP-1 cells or neutrophils. After 1 h incubation, THP-1 cells or neutrophils were washed three times with RPMI and incubated at 37°C. After 6 h incubation, THP-1 cells or neutrophils were fixed in 4% paraformaldehyde, permeabilized and stained with anti-influenza NP antibody (Abcam, Ab20921, 1:100 dilution). The levels of phagocytosis were monitored by flow cytometry.
3
Influenza Virus Nucleoprotein Detection
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Infected cells were detected by flow cytometry using an anti-influenza A virus nucleoprotein 1 (FITC) antibody [431] (Abcam, Cambridge, UK: ab20921), as previously described (16) .
4
Dual Detection of Influenza A and Streptococcus pneumoniae
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Single or mixed infected cells were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized in PBS-0.1% Triton X-100, and incubated with PBS containing 1% BSA and 3% human serum to block non-specific binding. Mouse monoclonal anti-Influenza A Virus Nucleoprotein (#ab20921; Abcam, Cambridge, United Kingdom) and goat anti-mouse IgG H&L (AF-488, #A11029; Life Technologies) were used to detect IAV. Rabbit polyclonal anti-Streptococcus pneumoniae antibody (#ab20429; Abcam) and goat anti-rabbit IgG H&L (AF-647, #ab150083; Abcam) were used to detect SP. Cells were imaged using a Leica DMI 3000B microscope equipped for fluorescence imaging.
5
Quantifying Influenza A Virus Lung Infection
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The degree of IAV infection was determined by measuring the relative proportion of lung cells expressing influenza nuclear protein (NP) in lung homogenate from infected and control mice. Briefly the lungs from infected mice (or naïve controls) were homogenized to single cell suspension by pressing them through a 100m nylon mesh strainer and incubated with red blood cell lysis buffer (Roche #11814389001) for 5 minutes at 4C. Cell viability was assessed using Live/Dead stain (Thermofisher L23102), with monoclonal mouse antiinfluenza A virus nucleoprotein antibody conjugated to FITC (Abcam #ab20921) to determine infection. The percentage of live lung cells infected with IAV was determined by using flow cytometry (BC Epics XL-MCL flow cytometer) by gating on live cells expressing nucleoprotein on the FL1 FITC channel. We determined the percentage of lung cells expressing influenza NP; and therefore infected with influenza A virus.
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