Streptavidin and antibodies to B220, CD5, CD19, CD21, CD23, CD45.1, CD45.2 CD93, IgD, Igλ1, Igλ1,2,3, IgM, IgM[a], and IgM[b], were conjugated to biotin, APC/A647, APC-e780, FITC, PE, PE-Cy7, PerCP-Cy5.5, or Pacific Blue (Tonbo Biosciences, Biolegend, BD Biosciences, eBioscience). NP hapten conjugated to PE was from Biosearch Technologies. pErk Ab for intracellular staining (clone 194g2) was from Cell Signaling Technologies. Donkey anti-rabbit secondary Ab conjugated to APC was from Jackson Immunoresearch. Goat anti-mouse IgM F(ab’)2 stimulatory antibody was from Jackson Immunoresearch.
Np hapten conjugated to pe
Manufactured by Biosearch Technologies
The NP hapten conjugated to PE is a fluorescent labeling reagent used in various bioanalytical applications. The NP hapten is covalently attached to the fluorescent dye phycoerythrin (PE), providing a labeled molecule that can be detected and quantified.
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2 protocols using np hapten conjugated to pe
1
Multiparametric Flow Cytometry Analysis
2
Multiparameter Flow Cytometry Analysis
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Single-cell suspensions were resuspended in PBS 2% FCS and stained for flow cytometric analysis. Data were acquired with a LSR Fortessa (BD Biosciences) and analyzed using FlowJo software. Antibodies against the following molecules were used: CD19 (6D5), CD93 (AA4.1), CD23 (B3B4), CD1d (1B1), GL7 (GL-7), β1-integrin (HMb1-1), β2-integrin (M18/2), VCAM-1 (429), anti-rat PE to detect VCAM-1, and IgG2a isotype control were from eBioscience; CD21 (7G6), B220 (RA3-6B2), CD138 (281-2), CD5 (53-7.3), IgM (R6-60.2), Igλ (R26-46), Fas (Jo2), Gr1 (RB6-8C5), and α4-integrin (9C10) from BD. αL-integrin (M17/4) and CD11b (M1/70) were from BioLegend. NP hapten conjugated to PE was from Biosearch Technologies. For detecting intracellular phosphorylated proteins, cells were stained for 20 min with surface markers and then cells were fixed and permeabilized with Cytofix/Cytoperm solution according to the protocol’s instructions (catalog No. 554723; BD Biosciences). Cells were incubated overnight (ON) with the unconjugated rabbit anti-mouse p-Syk (C87C1; Cell Signaling Technology) and rabbit anti-mouse p-Lyn (Cell Signaling Technology) followed by AF488 or AF647-conjugated anti-rabbit secondary antibody. Anti-CD16/32 (93; BD) was used to block nonspecific binding.
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