Journal: Vaccines

Article Title: Effectiveness of Booster Dose of Anti SARS-CoV-2 BNT162b2 in Cirrhosis: Longitudinal Evaluation of Humoral and Cellular Response

doi: 10.3390/vaccines10081281

Figure Lengend Snippet: Schematic overview of experimental approach for the detection of B cells interacting with SARS-CoV-2 recombinant S protein by flow cytometry. ( A ) HEK-293T cells were transiently transfected with pcDNA3.1 vector, encoding the receptor binding domain (RBD) of SARS-CoV-2 protein S (spike) fused with sfGFP fluorescent marker. Afterwards, the recombinant SARS-CoV-2 protein was purified after 2 days from transfection and employed for the immunostaining of human PBMCs after red blood cell lysis. Initially, cells interacted with the recombinant SARS-CoV-2 proteins or sfGFP only as control. Subsequently after washing, cells were labelled with the reported B-cell panel of fluorophore-conjugated antibodies. ( B ) Flow cytometry plots of B cells from a representative sample after interaction with the recombinant sfGFP-tagged SARS-CoV-2 S/RBD protein, only sfGFP as control and following the blocking of binding with native unlabeled SARS-CoV-2 S/RBD protein. Plot of non-B cells from the same sample after interaction with the recombinant sfGFP-tagged SARS-CoV-2 S/RBD protein is also reported. B cells were determined in the CD19+CD3- cell fraction. Boolean gate was applied to identify non-B cells using the FlowJo (Becton Dickinson) software. ( C ) Overview of gating strategy for identifying the different B-cell subsets in peripheral blood mononuclear cells (PBMCs). Fluorescence minus one (FMO) controls were used to set up all gates. Singlets were initially discriminated on SSC-H and SSC-A, followed by the exclusion of non-viable cells with Live/Dead far-red fluorescent DNA dye and the identification of CD45+ cell fraction. B cells were identified as CD3-CD19+ and plasma B cells were differentiated as CD38 high within the subset of B cells.

Article Snippet: After 20 min of cell protein incubation at room temperature, cells were washed with PBS and then stained with an anti-human CD45 eFluor 506-conjugated antibody (BDBioscience), an anti-human CD19 PE-conjugated antibody (BDBioscience), an anti-human CD38 PECy5-conjugated antibody (BDBioscience) and an anti-human CD3 SB436–conjugated antibody (BDBioscience, Franklin Lakes, NJ, USA).

Techniques: Recombinant, Flow Cytometry, Transfection, Plasmid Preparation, Binding Assay, Marker, Purification, Immunostaining, Lysis, Blocking Assay, Software, Fluorescence