Cell lysis buffer

Samples of the spleen and the blood were collected for the flow cytometry from anesthetized mice at 3 d post B-cell depletion, 24 d after B-cell depletion/ 21 d (3 w) after AAV-PHP.eB injection, and 4 d of the NK-cell depletion. While the blood sample was simply collected, the removed spleen was cut into pieces and ground. Then, the samples were passed through 70 microns (um) nylon mesh for single-cell suspensions, followed by centrifugation at 300 × g for 10 min. Red blood cells in the sample were lysed with cell lysis buffer (559759, BD Pharmingen, San Jose, CA, USA), and the remaining cells were washed three times and counted (1×10⁶ cells in a 100 µl for staining).
For B-cell staining, the antigens on the cell surface were labeled with anti-B220 (1 µg/100 µl, APC, BD Biosciences, San Jose, CA, USA), anti-CD3 (1 µg/100 µl, BV510, BD Biosciences), and anti-CD45 (1 µg/100 µl, Percp-cy5.5, BD Biosciences) at RT for 50 min. For NK-cell staining, cells were incubated with anti-NK49b (1 µg/100 µl, FITC, BD Biosciences), anti-CD3, and anti-CD45. Stained cells were collected with BD Verse (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

For tetramer staining, lithium-heparin anticoagulated whole blood samples were stained with anti-mouse CD3, anti-mouse CD8 antibodies (BioLegend) and MHC-I H-2K^d HIV Gag Tetramer-AMQMLKETI-APC (MBL) for 30 min at 4 °C. Red blood cells were depleted by cell lysis buffer (BD Biosciences). Cells were then washed twice with PBS containing 2% FBS, and re-suspended in 1% paraformaldehyde. Samples were acquired using a BD FACSCanto II cytometer (BD Biosciences) and the data were analyzed with FlowJo software (Tree Star).