Ia 1 e af647

Manufactured by BioLegend

The IA/I-E AF647 product is a fluorescent-labeled antibody that targets the I-A and I-E molecules on the surface of antigen-presenting cells. It can be used for the detection and analysis of these cell surface markers in flow cytometry applications.

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2 protocols using ia 1 e af647

Quantifying Virus-Infected Immune Cells

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Cells were incubated for 30 min on ice in 100 μl Hank’s balanced salt solution (Thermo Fisher) + 2% FBS with Fc block (1:100 dilution, clone 2.4G2; BD Biosciences) along with the following antibodies (all from BD Biosciences): CD45 APC-Cy7 (1:500, clone 30-F11), CD11b AF488 (1:500, clone M1/70), CD80 BV421 (1:200, clone 16-1OA1), and CD86 PE-Cy7 (1:500, clone GL1). IA/I-E AF647 (1:500, clone M5/114.15.2; BioLegend). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) according to manufacturer’s instructions. Cells were then incubated with an anti-RVFV antibody (kindly provided by Dr. Robert Tesh and the World Reference Center of Emerging Viruses and Arboviruses) at 1:500 dilution, followed by goat anti-mouse PE secondary antibody (1:1000, Santa Cruz biotechnology). Flow cytometry was performed using a FACSAria Fusion and data were analyzed using FlowJo software. Microglia, other myeloid lineage, and lymphocytes were resolved using CD45 and CD11b expression, with microglia identified as CD45^int CD11b^int, other myeloid as CD45^hi CD11b^hi, and lymphocytes as CD45^hi CD11b^– as previously described [65 (link)].

Hum N.R., Bourguet F.A., Sebastian A., Lam D., Phillips A.M., Sanchez K.R., Rasley A., Loots G.G, & Weilhammer D.R. (2022). MAVS mediates a protective immune response in the brain to Rift Valley fever virus. PLoS Pathogens, 18(5), e1010231.

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Neonatal Lung Immune Cell Profiling

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Neonatal lung single‐cell suspensions were used for all immunostaining. Panels of monoclonal antibodies (purchased from BD Bioscience unless otherwise stated) were developed to enable phenotypic characterisation of leucocytes of myeloid: CD45‐PerCP (clone 30‐F11), CD11b‐v500 (clone M1/70), CD11c‐AF700 (clone HL3), CD19‐BV786 (clone 1D3), CD103‐PE (clone M290), CD301‐PE‐Cy7 (clone MGL1/MGL2; BioLegend), F4/80‐FITC (clone BM8; BioLegend, San Diego), Ly6G/C‐APC‐Cy7 (clone RB6‐8C5), I‐A/I‐E‐AF647 (clone M5/114.14.2) and B220/CD45R‐PE‐CF594 (clone RA3‐6B2) and lymphoid: CD45‐PerCP (clone 30‐F11), NKp46‐PE‐Cy7 (clone 29A1.4; BioLegend), CD19‐BV786 (clone 1D3), CD3‐FITC (clone 17A2), CD4‐v500 (clone RM4‐5), CD8α‐BV650 (clone 53‐6.7), CD25‐APC‐Cy7 (clone PC61) and Foxp3‐PE (clone FJK‐16s) lineages. Intracellular staining for Foxp3 was performed using an intracellular Foxp3/Transcription Factor Staining Buffer Kit (eBioscience, San Diego). All samples were kept as individuals. Immune cell phenotypic characterisation was performed using the FlowJo software (version 10.6.1; BD Bioscience). Fluorescent minus one staining controls were used for all panels where necessary. Flow cytometry data quality was based on primary time gates to ensure appropriate laser delay (pre‐determined by automated CS&T) during sample acquisition.

Lauzon‐Joset J., Mincham K.T., Scott N.M., Khandan Y., Stumbles P.A., Holt P.G, & Strickland D.H. (2021). Protection against neonatal respiratory viral infection via maternal treatment during pregnancy with the benign immune training agent OM‐85. Clinical & Translational Immunology, 10(7), e1303.

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