Lsrfortessa ow cytometer

Cells were resuspended with 75% alcohol at -20°C overnight. After that cells were incubated with 1 mg/ml RNase A stock solution for 30min at 37°C, and stained with 100 μg/ml propidium iodide (BD) for 5min at room temperature, and assessed on an LSRFortessa ow cytometer (BD). Data analysis were conducted using Mod t software.

The mitochondrial membrane potential (MMP) was monitored using DiOC 6 , as described previously (32) .
For each condition, 1 × 10 6 cells were incubated with 1 mL of DePsipher solution (Trevigen, Gaithersburg, MD, USA), which uses a cationic dye (5,5'6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide). After incubation for 20 min at 37 °C in a 5% CO 2 incubator, cells were washed with 1 mL of prewarmed 1X Reaction Buffer with Stabiliser Solution and subsequently analysed using an LSR Fortessa ow cytometer (488 nm argon laser) and the FACSuite software (BD Biosciences).

Apoptosis evaluation was performed by an Annexin V binding assay using LSR Fortessa ow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were treated with various concentrations of venetoclax with or without ATO for 48 h and resuspended in Annexin V binding buffer. They were then incubated with Annexin V-FITC (BD Biosciences) and propidium iodide (PI) or 7-AAD (Beckman Coulter, Brea, CA, USA) for 15 min before ow cytometry analysis. To examine the apoptosis in the CD34 + CD38 -cell fraction, cells were stained with anti-CD34-APC (BD Biosciences), anti-CD38-PE (BD Biosciences), and 7-AAD (Beckman Coulter) for 30 min. The labelled cells were subsequently resuspended in Annexin V binding buffer and incubated with Annexin V-FITC (BD Pharmingen) for 15 min before ow cytometry analysis. Data were analysed using the FACSuite software (BD Biosciences).

Cells were resuspended with 75% alcohol at -20°C overnight. After that cells were incubated with 1 mg/ml RNase A stock solution for 30min at 37°C, and stained with 100 μg/ml propidium iodide (BD) for 5min at room temperature, and assessed on an LSRFortessa ow cytometer (BD). Data analysis were conducted using Mod t software.

Mice were euthanized with CO2 and transcardially perfused with ice-cold PBS (25) . Lateral ventricle choroid plexus (LVCP) were carefully isolated under a microscope and placed in 1.5 ml tube with ice-cold PBS. CL or IL ChP were enzymatically digested in collagenase type IV (400 U/ml in PBS, 45 min, 37° C), and then triturated several times using a pipette (28). Cells were stained with antibodies (Supplementary Table 1) for 20 min at 4°C in the dark. Samples were acquired using an LSRFortessa ow cytometer (BD Biosciences, USA) equipped with FACS Diva software and a minimum of 30,000 events for each sample was recorded. Data were analyzed using the Flow Jo (BD Biosciences, USA) software.

Lysed cells were acquired and subsequently stained for 30 min at 4 °C with the following uoresceinconjugated monoclonal antibodies: CD3-PE (Bio-Legend, San Diego, CA, USA), CD4-APC (eBioscience, San Diego, CA, USA), CD8a-Percp/Cy5.5 (eBioscience), CD45RO-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CCR7-APC/Cy7 (BioLegend). The T cell subsets were de ned as previous study reported [10] : Naive T cells being CCR7 + and CD45RO -; central memory T cells as CD45RO + and CCR7 + ; effector memory T cells as CD45RO + and CCR7 -, and effector memory RA (EMRA) T cells as CD45RO -and CCR7 -(Figure S1). A total of 200,000 events were acquired by the BD LSRFortessa™ ow cytometer (BD Bioscience, San Jose, CA, USA). The data analysis was carried out with Flowjo v10.1 Software (Tree Star, Ashland, OR). The absolute number of each T cell subset was calculated as follows: (percentage of each cell population among total lymphocytes)×(total lymphocytes count)/100.