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), GL7 (GL-7), CD184 (CXCR4-2B11) from eBioscience; CD21 (7G6), B220 (RA3-6B2), CD138 (281-2), CD45.1 (A20), CD45.2 (104), IgM (R6-60.2), Fas (Jo2) from BD. Anti-CD29 (9EG7) in combination with secondary anti-rat PE antibody (both BD) was used to detect the active conformation state of β1-integrin. Anti-CD29 (HMb1-1) antibody was used to detect the total (pan) β1-integrin (eBioscience). IgG1 biotinylated antibody (Southern Biotech) was conjugated with SA-BV421 (Biolegend). NP-PE antibody was from Biosearch Tech. Anti-CD16/32 (93) (BD) was used to block nonspecific binding.
Anti cd16 32 93
Manufactured by BD
Anti-CD16/32 (93) is a laboratory reagent that binds to the CD16 and CD32 receptors on the surface of immune cells. It is commonly used in flow cytometry and other cell-based assays to label and identify specific cell populations.
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Multiparameter flow cytometry analysis
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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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