Automacs rinsing solution

Thirty hindbrains of HH18 chick embryos were harvested and dissociated into single cells using collagenase type 4 (200 units/ml, Worthington 47B9407). Cells were then centrifuged at 600 g for 10 min, washed in PBS, then centrifuged again. Next, cells were incubated with mouse anti-CSPG antibody (c8053, Sigma-Aldrich) diluted 1:50 in MACS BSA Stock Solution and autoMACS Rinsing Solution (1:20, Miltenyi Biotec) for 75 min at room temperature. Next, cells were centrifuged and washed in PBS twice, then incubated with anti-mouse Alexa-Fluor 488 antibody (1:200, Thermo Fisher Scientific) in autoMACS Running Buffer (Miltenyi Biotec) for 30 min at room temperature. Cells were then washed, centrifuged, resuspended and kept in hESC media overnight at 4°C. Next, cells were washed with autoMACS Running Buffer and stained with DAPI (1:200 in autoMACS Running Buffer) for 5 min at room temperature, then washed again. 1×10⁷ cells/ml were passed to FACS tubes and sorted using an ARIA III FACS (BD Biosciences) into 1 ml autoMACS Running Buffer. The gating was set according to size and granularity using FSC and SSC to capture singlets and remove debris. The cut-off for sorting the positive (CSPG⁺) and negative (CSPG⁻) cells was based on Alexa-Fluor 488 stained or unstained cells, appropriately, with the exclusion of dead and/or damaged DAPI⁺ cells, chosen by manual gating.

SIV Env-specific CD4 T cells were characterized as previously described (33 (link)). Briefly, 1 × 10⁶ to 2 × 10⁶ splenic and iliac LN mononuclear cells were stimulated for 5 h with 1 μg/ml of an overlapping SIV_mac239 Env peptide pool (NIH AIDS Reagent Program) in the presence of GolgiStop and GolgiPlug (BD Biosciences). Cells were washed three times in autoMACS rinsing solution (Miltenyi Biotec) and stained with vital exclusion dye (Life Technologies) for 10 min at 4°C. Cells were again washed and treated with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C. All subsequent washes and stainings were performed by using BD Perm/Wash (BD Biosciences). Cells were incubated with an interleukin-21 (IL-21) receptor (IL-21R)/Fc fusion protein (R&D Systems) for 30 min at 4°C, washed three times, and stained with goat anti-human Fcγ antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Cells were subsequently washed three times and stained with anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD44 (IM7), anti-gamma interferon (IFN-γ) (XMG1.2), and anti-IL-2 (JES6-5H4) for 30 min at 4°C. Samples were washed three additional times, fixed in 2% formaldehyde, and stored at 4°C. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed by using FlowJo v9.8.3 (TreeStar).

Spleens were homogenized and filtered through 40μm nylon strainer to obtain a single cell suspension. Cells were resuspended in autoMACS Rinsing Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACS BSA stock solution (Miltenyi Biotec). Small portion of cells were first stained with human CD45 clone HI30, human CD3 clone UCHT1, human CD20 clone 2H7, and mouse CD45 clone 30-F11. Cell numbers in each spleen sample were measured with LSR Fortessa Cell Analyzer (BD Biosciences, San Jose, CA) and data were analysed with FlowJo software (Tree Star, Inc., San Carlos, CA). Based on the cell number, whole spleen cell suspension was incubated with human CD45 antibody (Clone HI30). Human CD45+ cells were collected in PBS by FACS Aria (BD Biosciences). To obtain enough cell number for proteomic analysis, samples were pooled based on the number of collected cells by flow cytometry. After pooling samples, total 21samples (0 Gy, n = 7; 1 Gy, n = 7; 2 Gy, n = 7) were prepared for proteomic analysis. Cell pellets were frozen and stored at −80 °C until use.