Propidium iodide p4864

Blood was obtained from healthy human donors before and on day 6 or 7 after vaccine immunization with the seasonal influenza Fluzone (year 2018). Samples were obtained after written informed consent was provided and ethical approval granted from the Western Institutional Review Board. Blood samples were diluted in phosphate-buffered saline (PBS) at a 1:1 (volume/volume) ratio and layered on top of a Ficoll-Paque-PLUS medium cushion (density 1.077 g/ml, GE Healthcare Life Sciences). Samples were centrifuged for 30 min at room temperature at 400 g with a soft stop. The interface layer containing peripheral blood mononuclear cells was collected and resuspended in FACS staining buffer (PBS, 0.5% BSA, and 2 mM EDTA) for marker staining and cell sorting. Single-cell suspensions at 5 × 10⁷ cells/ml were stained with a cocktail of fluorochrome conjugated human antibodies (BD Biosciences, San Jose, CA), anti-human IgG APC, anti-human CD20 PE Cy7, anti-human CD4 APC Cy7, Propidium iodide (PI) P4864 (Sigma-Aldrich, St. Louis, MO). IgG^pos B cells were enriched using PE-dump channel cocktail antibodies to exclude granulocyte, monocyte/macrophage, dendritic, NK, and CD8 cell populations. IgG^pos B cells (100,000 cells from each donor) were sorted on a FACSAria II cell sorter (BD Biosciences, San Jose, CA) and collected for scBCR-seq (Supplementary Fig. 15).

Cells were plated on glass coverslips, starved overnight and treated as described in the text. Cells were fixed using 4% paraformaldehyde/PBS for 15 min and permeabilized with 0.5% NP-40 for 5 min. Next, cells were blocked using 3% BSA, 0.01% TritonX-100 in PBS for 20 min. Furthermore, cells were incubated with anti-EGFR, overnight at 4°C followed by 3x washes with PBS/ 0.01% TritonX-100 and incubation with the AlexaFluor^® 488-labeled (#A11034 Invitrogen, Carlsbad, CA, USA) for 60 min at room temperature. After 3x washes with PBS/ 0.01% TritonX-100, nuclei were stained with 6-diamidino-2- phenylindole (DAPI #D9542) or propidium iodide (PI #P4864) 1μg/ml (Sigma Aldrich) for 20 min. The coverslips were mounted with fluorescent mounting medium (#S3023 Dako, Glostrup, Denmark) on microscope slides. Cells were analyzed with a confocal laser scanning microscope Leica SP5. Images for documenting EGFR nuclear translocation and colocalization with Caveolin-1 were acquired in the middle section of the nuclei with 63x magnification.

For apoptosis analysis, cells were stained with Annexin V-PE and 7-AAD (AP104, Multi Sciences) and evaluated by flow cytometry according to the manufacturer's protocol. Briefly, 1 × 10⁶ cells were washed twice with PBS and stained with 5 μl Annexin V-PE and 10 μl 7-AAD in 1× binding buffer for 15 min at room temperature in the dark. Apoptotic cells were determined using a Beckman-Coulter Flow Cytometry FC500. Both early (Annexin V-positive/7-AAD-negative) and late (Annexin V-positive/7-AAD-positive) apoptotic cells were included when assessing cell death.
For cell cycle analysis, samples were harvested, washed twice in PBS, and then fixed in ice-cold 70% ethanol at −20°C overnight. The fixed cells were treated with RNase A (R4875, Sigma-Aldrich) for 30 min at room temperature before the addition of 5 μl/ml propidium iodide (PI, P4864, Sigma-Aldrich) for 10 min in the dark and analysis by flow cytometry.