Quantifying Tat-induced Multinucleation in Cells

Jurkat-Tat101 and Jurkat-Tat72 or PBMCs transiently transfected with Tat101 were adhered on PolyPrep slides (Sigma-Aldrich) and fixed with 2% paraformaldehyde in PBS1X. Immunofluorescence assays were performed as previously described.[8 (link)] The number of giant cells with multi-lobed nuclei was calculated by acquiring 60 fields—containing an average number of cells close to 40—of each cell type with a Leica DMI 4000B Inverted Microscope (Leica Microsystems) after staining the cells with a monoclonal antibody against α-tubulin (Sigma-Aldrich), followed by secondary antibody conjugated to Alexa 546 (Thermofisher). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Images were obtained with a Leica DMI 4000B Inverted Microscope (Leica Microsystems). The percentage of cells showing a giant, multi-lobed nuclear morphology was calculated considering the total number of cells. Data were normalized accordingly to the levels found in control cells. The diameter of the cells was measured by using LAS AF software (Leica Microsystems).