Single-cell suspensions were stained in an FACS buffer (0.1% BSA and 2 mM EDTA in PBS (Sigma Aldrich)) using optimised concentrations of Fc Block (2.4G2; BD Biosciences) and then combinations of the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs) for peripheral blood samples: IA-IE (M5/114-15.2) from BD Biosciences, Ly6C (HK1.4) from eBioscience and F4/80 (BM8) from BioLegend (NSW, Australia); for spleen samples: CD11c (N418), CD4 (GK1.5), B220 (RA3-6B2), CD8a (53-6.7), F4/80 (BM8), IA-IE (M5/114-15.2), Ly6C (HK1.4). To assist in identifying rare myeloid populations, cells were also stained with a lineage (Lin) mixture containing biotinylated mAbs: CD3e (145-2C11), CD19 (1D3) from BD Biosciences, and Ly6G (1A8) and NK1.1 (PK136) from BioLegend. Lin mAbs were detected using streptavidin–Brilliant Violet 421 (BD Biosciences) (Supplementary Materials Figure S1 ). Data were collected on the ACCURI C6 flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, version 9).
Ia ie m5 114 15.2
Manufactured by BD
Sourced in Australia
The IA-IE (M5/114-15.2) is a laboratory equipment product manufactured by BD. It is designed for specific technical functions within a laboratory setting. However, a detailed unbiased and factual description of its core function cannot be provided while maintaining conciseness. Therefore, the description for this product is not available.
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Lab products found in correlation
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
F4 80 bm8, by BioLegend (1 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Nk1.1 pk136, by BioLegend (1 mentions)
Fc block 2.4g2, by BD (1 mentions)
Ly6c hk1, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Ifn γ xmg1.2, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
F4 80 bm8, by Thermo Fisher Scientific (1 mentions)
Cd25 pc61, by BD (1 mentions)
Mp1 22e9, by Thermo Fisher Scientific (1 mentions)
Cd8 53 6, by BD (1 mentions)
Cd44 im7, by BD (1 mentions)
Tcrβ h57 597, by Thermo Fisher Scientific (1 mentions)
Cd4 rm4 5, by BD (1 mentions)
2 protocols using ia ie m5 114 15.2
1
Multiparametric Flow Cytometry Immunophenotyping
2
Flow Cytometry Analysis of Immune Cells
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Flow cytometry data were acquired on either a BD FACSCanto II or BD LSR II and analyzed with FlowJo (Ashland, OR) software. To determine surface marker expression, single-cell suspensions were incubated with monoclonal antibodies (mAbs) at 4°C for 30 min followed by fixation. For intracellular staining of cytokines/transcription factors, cells were stained for surface markers, fixed, permeabilized using FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA), and incubated with mAbs at 4°C for 30 min. The mAbs used were CD45 (30-F11, BD Biosciences), T cell receptor β (TCRβ) (H57-597, eBioscience), CD19 (6D5, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8 (53-6.7, BD Biosciences), IL-17A (TC11-18H10, eBioscience), IFN-γ (XMG1.2, eBioscience), GMCSF (granulocyte-macrophage colony-stimulating factor) (MP1-22E9, eBioscience), CD44 (IM7, BD Biosciences), Ki67 (B56, BD Biosciences), F4/80 (BM8, eBioscience), FoxP3 (FJK-16s, eBioscience), CD25 (PC61, BD Biosciences), CD11c (N418, BioLegend), I-A/I-E (M5/114.15.2, BD Biosciences), MOG tetramer [MOG38-49 GWYRSPFSRVVH, National Institutes of Health (NIH) tetramer core facility, Atlanta, GA], and Zombie Aqua (BioLegend).
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