The total ROS production was detected by H2DCFDA, according to the manufacturer's instructions (Invitrogen). Cells were quantified by flow cytometry following manufacturer's instructions. Data were analysed by FlowJo (Tree Star, Inc.). For detection of neutrophils or intracellular TNF‐α, cells were stained with APC‐labelled rat anti‐mouse CD45 antibody (1:100; BD Biosciences, Franklin Lakes, NJ, USA), PerCP‐Cy5.5‐labelled rat anti‐mouse CD11b antibody (1:100; BD Biosciences) and FITC‐labelled rat anti‐mouse Ly6G antibody (1:100; BD Biosciences) for 30 minutes in PBS at 4°C and then washed twice with PBS. Then, the cells were fixed in 4% paraformaldehyde solution for 20 minutes, permeabilized with 0.5% Tritox‐100 for 30 minutes at 4°C and washed with PBS. Next, the cells were stained with a PE‐labelled rat anti‐mouse TNF‐α antibody (1:100; BD Biosciences) for 2 hours at 4°C and then washed. Data acquisition was performed on an Aria III flow cytometer, and the results were analysed by Flowjo, Version 7.6.1; Treestar, Ashland, OR, USA.
Aria 3 flow cytometer
Manufactured by Tree Star
Sourced in United States
The Aria III flow cytometer is a high-performance instrument designed for advanced cell analysis and sorting. It utilizes cutting-edge technology to provide accurate and reliable data on various cell populations, their characteristics, and their behavior. The core function of the Aria III is to enable the detection, identification, and separation of different cell types within a heterogeneous sample.
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Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Apc labelled rat anti mouse cd45 antibody, by BD (1 mentions)
Percp cy5.5 labelled rat anti mouse cd11b antibody, by BD (1 mentions)
Pe labelled rat anti mouse tnf α antibody, by BD (1 mentions)
Cd11c apc, by BD (1 mentions)
Cd44 apc, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Cd8 pe, by BD (1 mentions)
Cd19 fitc, by BioLegend (1 mentions)
Cd38 bv711, by BD (1 mentions)
Lsr fortessa, by Tree Star (1 mentions)
Percp cy5.5 cd8, by BD (1 mentions)
Percp cy5.5 cd3, by BD (1 mentions)
Live dead aqua 525, by Thermo Fisher Scientific (1 mentions)
2 protocols using aria 3 flow cytometer
1
Quantifying Cellular ROS and Inflammatory Markers
2
Influenza NP-specific CD8+ T cell analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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MHC‐I tetramer targeting the immunodominant epitope of the influenza nucleoprotein (DbNP366–374 – ASNENMETM, DbPA224–233 – SSLENFRAYV) was produced in‐house and conjugated to streptavidin‐APC/PE (Life Technologies Australia Pty Ltd) at a 1:250 dilution at room temperature for 1h. Cells were stained with combinations of fluorochrome‐conjugated antibodies: PerCP‐Cy5.5‐CD3 (#551163), PE‐CD8 (#561095), BV421‐I‐Ab (#562928), APC‐CD44 (#553133), FITC‐CD44 (#553133), BV711‐CD38 (#740697), PerCP‐Cy5.5‐CD8 (#551162), APC/eF780‐CD62L (#47‐0621‐82), APC‐CD11c (#17011481), PE‐Cy7‐TCR‐va2 (#560624) from BD Biosciences, USA; and PE‐Cy7‐CD38 (#102718), BV785‐PD‐1 (#329908), APC‐Cy7‐CD45.1 (#110716), FITC‐I‐Ab (#116406), Pacific Blue‐I‐Ab (#116422), FITC‐CD19 (#115506) from Biolegend, USA. AF700‐CD3 (#56003382) was purchased from Invitrogen, USA.
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
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