Live dead aqua viability stain

Custom-made AD-6 (CMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYV with a GGC tag) and AD-6.4 (REFNSYKQRVKYVED with a GGC tag) were purchased from Peptide 2.0 US. Custom-made affinity-purified anti-AD-6 rabbit IgG was generated by GenScript.
A Recombinant gB fragment was purchased (Abcam; ab43040) and the vaccine gB was a kind gift of Sanofi Pasteur.
Anti-IE CMV (6F8.2, MAB8131, Millipore; 1:2000 dilution). Goat anti-mouse IgG (H + L)-AlexaFluor 568 (A11004, Invitrogen, 1:2000 dilution) and Live/Dead Aqua viability stain (Life Technologies, 1:1000 dilution). The ITC88 antibody was purchased from CreativeLabs.

HFF cells were infected with IE2-GFP virus (MOI:5) cells and fixed with 2% PFA 3 days post infection. Cells were blocked (2% BSA) and stained with Live/Dead Aqua viability stain (Life Technologies, 1:1000 dilution), and an anti-AD-6 antibody at 50 μg/mL. Following 30 minutes of incubation, cells were washed and stained with PE-conjugated anti-rabbit IgG (L42018, Invitrogen; 1 μg/ml) and an APC-Cy7 conjugated HLA-A-C MHC class I antibody (clone W6/32Biolegend, 1:50 dilution) for 30 minutes, followed by washing. Infected cells were identified by MHC class I downregulation. Samples were acquired using an LSR Fortessa II, BD FASCDiva software and analysed with FlowJo v10.

Ninety days after the initial FTG, mice were anesthetized with Avertin (250 mg/kg, i.p.) and blood was collected by cardiac puncture using a heparinized syringe. Each mouse was perfused transcardially using ice cold PBS solution and the brain was removed. The right hemisphere was homogenized and incubated in RPMI-1640 (Corning) containing 1 mg/ml of collagenase/dispase (Roche) and 10 mg/ml of DNase I (Roche) for 45 min at 37°C. The cell suspension was filtrated through a 70-μm cell strainer, washed and applied to a Percoll (GE Healthcare) gradients of 30% and 70% using the centrifugation at 500 g for 20 min. The interphase between the gradients was collected. Cells were stained with anti-CD45 (clone: 30-F11, BioLegend 103139), anti-CD4 (clone: RM4-5, BioLegend 100516), anti-CD8α (clone: 53–6.7, BioLegend 100737) and anti-TCRγδ (clone: GL3, BioLegend 118124). An amine reactive Live/Dead Aqua viability stain (Invitrogen L34966) was used to identify only live cells. Fluorescence-minus-one controls were used to distinguish positively stained cells for each antibody. The cells were analyzed using CytoFLEX (BECKMAN COULTER). The data were analyzed using FlowJo software (Tree Star, Inc.).