Pe donkey anti rabbit igg antibody

Four days after infection, T cells were harvested and washed once with PBS, stained with rabbit anti-FLT3L antibody (Abcam, USA) for 1 h at 4 °C, and washed twice. Then PE donkey anti-rabbit IgG antibody (Biolegend, USA) was added, incubated at 4 °C for 30 min, and analyzed by flow cytometry using CantoII flow cytometer (BD Biosciences, San Jose, CA, USA) [14 (link)]. For T cell phenotype analysis, T cells were harvested 7 days after infection and washed once with PBS, stained with anti-CD4-PE/Cy7 (Biolegend, USA), anti-CD8-PerCP-Cy5.5 (Biolegend, USA), anti-CCR7-PE (Biolegend, USA), and anti-CD45RA-Pacific Blue (Biolegend USA) 30 min at 4 °C, then washed and resuspended in PBS for flow cytometry analysis [15 (link)].

CFPAC parental and HLA-A3 KO lines were harvested, washed with PBS, and resuspended in ice-cold flow cytometry staining buffer (PBS, 0.5% BSA, 2 mM, EDTA, 0.1% sodium azide) at a concentration of 10 × 10⁶ cells/mL. Next, 10 µL of precipitated phage (10¹³ titer) was applied to 100 µL of both cells and incubated on ice for 15 min. After washing 3X with staining buffer, cell pellets were resuspended to same concentration (10 × 10⁶ cells/mL) and stained with rabbit anti-M13 polyclonal antibody (Novus Biologicals, Littleton, CO) at 1:100 final dilution on ice for 15 min. After washing 3X again, cell pellets were resuspended and stained with PE donkey anti-rabbit IgG antibody (Biolegend, San Diego, CA) at 1:100 final dilution for 15 min on ice. After a final wash 3X, stained CFPAC cells were analyzed using a LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ) to measure mean fluorescence intensity. For gating, any PE signal above background in unstained cells was considered positive (Supplementary Fig. 6).

A549 cells were transduced with the multiplicity of infection (MOI) of 10 for the relevant hAd19a/64 vaccine. Twenty-four hours later, cells were incubated with 15 µg/mL of primary rabbit anti-human HERV polyclonal antibody (PA5-22819; Invitrogen™, Waltham, MA, USA) in FACS buffer (PBS containing 1% BSA and 0.1% NaN₃) for 1 h at 4 °C. This antibody targets the HERV-W Env SU region. Next, cells were stained with secondary phycoerythrin (PE) donkey anti-rabbit IgG antibody (406421; BioLegend^®, San Diego, CA, USA; 1:100) and eBioscience™ Fixable Viability Dye eFlour™ 780 (65-0865; Invitrogen™ Waltham, MA, USA; 1:1000) for 30 min at 4 °C. Finally, cells were fixated in 1% paraformaldehyde (PFA) for 15 min at 4 °C. Flow cytometry was run on the LSRFortessa™ 3-laser cell analyser (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analysed using FlowJo™ v10 analysis software and GraphPad Prism 9.