Computational protocol: Ex Vivo Analysis of Human Memory B Lymphocytes Specific for A and B Influenza Hemagglutinin by Polychromatic Flow-Cytometry

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Protocol publication

[…] Frozen PBMCs were thawed at 37°C in PBS containing 2.5 mM EDTA and 20 µg/mL DNAse (Sigma Aldrich). Samples were stained in tubes, each containing 107 PBMC, according to different protocols: i) For pre-saturation with mono-bulk vaccine antigens, PBMCs were first stained with Live/Dead Aqua (Invitrogen) diluted 1∶500 in 100 µl, for 20 min in the dark. Then 50 µl of PBS containing 20% rabbit serum were added for further 20 min at 4°C to saturate Fc receptors. After two washes in 2 ml of PBS, PBMCs pellets were dissolved in 10 µl of PBS containing 30 µg/ml of vaccine subunit antigens (or 60 µg/ml in case of staining with 2 antigen baits) and incubated at 4°C in PBS. After 15 min, 10 µl of PBS containing 30 µg/ml (approximately 4 micromoles) of each fluorochrome-conjugated rHA bait were added, together with 50 µl of a cocktail of pre-titrated amounts of anti-CD20 PrCPCy5.5 (Becton Dickinson, clone L27), anti-CD27 PE (Becton Dickinson, clone L128) and anti-hIgG PacificBlue (Jackson ImmunoRes) monoclonal antibodies 1% FBS for 1 hour at 4°C. After two washes with 2 ml of 1% FBS/PBS cells were diluted in 1 ml of 5 mM EDTA/PBS and acquired with the Canto II analyzer (Becton Dickinson, Pharmingen, San Diego, CA). ii) For neuraminidase pre-treatment, PBMCs pellets were diluted in 50 µl of PBS containing 0.1 to 5 M Type VIII neuraminidase from Clostridium perfringens (which cleaves α-2,3- α-2,6- and α-2,8-linked sialic-acid residues. # N5631 Sigma Aldrich), incubated for 30 min at 37°C and then washed 3 times with PBS. PBMCs were then stained as in i), but omitting the pre-incubation step with mono-bulk influenza antigens. iii) To saturate rHA binding sites for α2,6 linked sialic-acid residues, soluble sialopentasaccarides containing α2,6 linkage (provided by Dr. Paolo Costantino, Novartis VD) were incubated at 100-fold molar excess with labeled-rHA for 15 min at 4°C. PBMCs were then stained as in i), but omitting the pre-incubation step with mono-bulk influenza antigens. iv) For pre-incubation with the Sambucus Nigra lectin (Vector Laboratories), PBMCs were stained as in i) adding as saturation agent 100 µl of a solution of PBS containing 10 to 100 µg/ml of the lectin for 15 min at 4°C, instead of mono-bulk influenza antigens. Flow-cytometric analysis was performed using the FlowJo software (Treestar Inc.). For antigen-specific B-cell sorting, PBMCs from the same donor stained at 107/tube were then pooled, diluted in 1 ml of 5 mM EDTA/PBS and filtered through 30 µm cup filcon (Becton Dickinson). Samples were maintained on ice until sorting. Sorting was performed in high purity mode by a FACS Aria instrument (BD Biosciences, San Jose, CA) equipped with the BD FACSDiva v6.1.3 software using a 70 µm nozzle operating at 70 psi. Sorting gates were established in order to isolate brilliant HA+ CD20+ cells, unless differently specified. HA negative (HAneg) B cells and CD20 negative (CD20neg) cells were also sorted as negative controls or feeders for HA+ and HAneg B-cell cultures, respectively. […]

Pipeline specifications

Software tools FlowJo, BD FACSDiva, Nozzle
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Infection