The design and purification of fluorescently labelled recombinant HA probes with ablated sialic acid binding activity has been previously described (13 (link)). HA-specific B cells were identified within cryopreserved PBMC samples by co-staining with H1 (A/New Caledonia/20/1999) and H5 (A/Indonesia/05/2005) probes conjugated to streptavidin-PE or −APC (Life Technologies, New York, NY) respectively. B cells were stained with the following antibodies for sorting or phenotypic analysis: CD3-QD655, CD14-QD800, CD27-QD605 (Invitrogen), CD19-ECD (Beckman Coulter), IgM-Cy5.5-PerCP, IgG-FITC or −BV421, IgD-Cy7PE, CD38-Alexa Fluor 700, CD22-Cy5PE, CD24-Cy7PE, CXCR5-Ax488, CD20-Cy7APC (BD Pharmingen). Cell viability was assessed using Aqua Live/Dead amine-reactive dye (Invitrogen). The IGHV1-69 anti-idiotypic mouse monoclonal G6 was biotinylated and conjugated to Ax488 using standard procedures. 1 - 2 million events were collected on an LSR II instrument (BD Immunocytometry Systems) configured to detect 18 fluorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar). Where applicable, HA probes were incubated with scFv derived from influenza bNAb F10, or alternatively with VRC01 or VRC04 HIV-1 Env-specific scFv controls. Probes were incubated at room temperature with 10 μg/ml of scFv inhibitors for 1h prior to the addition of PBMCs.
Aqua live dead amine reactive dye
Manufactured by Thermo Fisher Scientific
The Aqua Live/Dead amine-reactive dye is a fluorescent dye used to detect viable cells. It selectively binds to primary amines on the cell surface, allowing for the identification of live and dead cells in a sample.
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Lab products found in correlation
Cd27 qd605, by Thermo Fisher Scientific (2 mentions)
Cd19 ecd, by Beckman Coulter (2 mentions)
Cd20 cy7apc, by BD (1 mentions)
Cd3 qd655, by Thermo Fisher Scientific (1 mentions)
Igd cy7pe, by BD (1 mentions)
Cd38 alexa fluor 700, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
F ab 2 fragment goat anti human iga, by Jackson ImmunoResearch (1 mentions)
3 protocols using aqua live dead amine reactive dye
1
Identification of HA-Specific B Cells
2
Isolation and Stimulation of Naïve B Cells
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Naïve B cell isolation kit II human (Miltenyi Biotec) was used to isolate naïve B cells from freshly isolated PBMCs according to the manufacturer’s instructions. One hundred thousand naïve B cells were stimulated with WT HA, or Y98F HA, each at 50 nM in RPMI containing 10% FBS. Control cells were stimulated with Anti-Ig [5 μg/ml, F(ab’)2 Fragment Goat Anti-human IgA + IgG + IgM (Jackson)]. After four days, the cells were washed in PBS and resuspended in 50 ul of PBS with Aqua Live/Dead amine-reactive dye (Invitrogen) (0.025 mg/ml for 2 minutes), quenched by addition of 50 ul 1% FBS in PBS, and then stained with a 1X cocktail of following antibodies: CD19 ECD (Beckman), CD27 QD605 (Invitrogen), IgM Cy5PerCP (BD Pharm) for 1 hour at 4 °C. The cells were then washed twice resuspended in 400 μl of PBS and then analyzed by flow cytometry.
3
Germline-reverted Stem Antibody Expression
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