Goat anti mouse igg bv421

293T cells were transfected with plasmid encoding the full-length rhLCV gB for 48 h. The transfected cells were trypsinized and resuspended by PBS and prepared with 10⁶ cells per test. The cells were stained with antibodies at 4°C for 30 min. After two washes, 293T cells were incubated with goat anti-mouse IgG BV421 (BioLegend) and anti-human IgG BV421 (BD Biosciences) at 4°C for 30 min in the dark. Stained cells were analyzed by flow cytometry (FCM) on an LSRFortessaX-20 instrument (BD Biosciences) and the data were evaluated using FlowJo software X 10.0.7 (BD Biosciences).

The plasmid of the full-length gp42 was transfected into 293T cells using PEI with a mass ratio of 1:3. The cells were trypsinized and resuspended by PBS and prepared by 10⁶ cells per test. The cells were stained with mAbs and negative control antibody 72A1 at 4 °C for 30 min. The cells were washed twice and incubated with goat anti-mouse IgG BV421 (Biolegend, San Diego, CA, USA) at 4 °C for 30 min in the dark. Stained cells were analyzed by flow cytometry on an LSRFortessaX-20 instrument (BD Biosciences, Franklin Lakes, NJ, USA) and the data were evaluated using FlowJo software X 10.0.7 (BD Biosciences, Franklin Lakes, NJ, USA).

Before sacrifice, three animals from each group were infused i.v. with 10 ml of 3.24 mg/ml purified CD3 Ab (clone PPT3) and sacrificed 10 min later. Lymphocytes were isolated and stained ex vivo with anti-mouse Ig-FITC (SouthernBiotech), which labels the circulating intravascular cells. The cells are washed, and normal mouse serum is then added to block any remaining binding capacity of the anti–Ig-FITC. The cells are then washed again and incubated with CD3 Ab labeled with PeCy5 (Abcam). This will bind unsaturated sites of the circulating cells, which are therefore double labeled, as well as all the sites on the T_RM that are not accessible to the CD3 Ab, given i.v. T_RM is therefore single labeled with PeCy5.
To allow intracytoplasmic staining of T_RM, the i.v. CD3 was detected with goat anti-mouse IgG BV421 (BioLegend) and blocked using normal mouse serum as above. Surface markers used were CD3ε-biotin PPT3 (Abcam), CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (all BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Biotinylated CD3 was visualized with a streptavidin AF647 (BioLegend). Cells were permeabilized using Cytofix/Cytoperm before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa.