Cells were washed and incubated for 20 minutes at 4°C with staining buffer (2% FCS in PBS containing 0.02% NaN3). Subsequently, the cells were stained with fluorescein isothiocyanate (FITC)‐conjugated anti‐CD163 (ED2, Bio‐Rad), phycoerythrin (PE)‐conjugated anti‐CD86 (24F, BioLegend) and pacific blue‐conjugated anti‐CD45 (OX‐1, BioLegend) or PE‐conjugated anti‐CD3 (1F4, BioLegend) and pacific blue‐conjugated anti‐CD45. Then, the cells were fixed and permeabilised with a fixation/permeabilisation solution kit (BD Biosciences) for 1 hour at 4°C in the dark, stained intracellularly with Alexa Fluor 647‐conjugated anti‐CD68 (ED1, Bio‐Rad) and analysed on a FACSCanto II Flow Cytometer (BD Biosciences), followed by analysis with FlowJo (Tree Star, Inc.; OR, US).
Fixation permeabilisation solution kit
Manufactured by BD
Sourced in United States
The Fixation/permeabilisation solution kit is a set of laboratory reagents designed to facilitate the fixation and permeabilisation of cells. The kit contains solutions for preparing cells for further analysis, such as immunostaining or flow cytometry.
Automatically generated - may contain errors
2 protocols using fixation permeabilisation solution kit
1
Multicolor Flow Cytometry of Immune Cells
2
Multiparametric Flow Cytometry Staining
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For staining of surface antigens fresh or thawed cells were incubated 30 minutes at +4c with antibodies and with Live/dead Fixable Green Dead Cell Stain (at dilution of 1:500; ThermoFisher) that were diluted in in Brilliant Stain Buffer (BD Bicoscience). When applicable the cells were incubated with MR-1 tetramer (1:2500) in +37c for 45 minutes and washed before continuing to other surface markers. For staining of transcription factors and Ki67 the cells were permeabilized after surface staining with FoxP3 transcription factor staining set (eBioscience) for and with Fixation/Permeabilisation Solution kit (BD Bioscience) for detection of intracellular cytokines as instructed by manufacturer. The samples were run using LSR Fortessa (BD Biosciences) and analyzed with FlowJo (BD Biosciences, LLC). The antibodies used in the study are shown in Supplementary Table X. Optimal concentration for antibodies was titrated with live PBMCs.
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