Lsrii special order flow cytometer

Splenocytes from immunized mice were isolated 2 weeks, 1 month or 4 months after the last immunization, plated at 1–2 × 10⁶ cells/well in 96-well plates, and stimulated with anti-CD28 and anti-CD49d (2 μg/ml each, BD Biosciences), EsxAB, Hla_H35L, FhuD2 or Csa1A (30 μg/ml), or with a combination of antigens (4C-Staph) containing EsxAB, Hla_H35L, FhuD2, and Csa1A (10 μg/ml each) at 37°C for 16–18 h, in the presence of Brefeldin A (5 μg/ml) for the last 4 h. The cells were then stained with Live/Dead Yellow (Invitrogen), fixed, and permeabilized with Cytofix/Cytoperm (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT, stained with fluorochrome-conjugated mAbs: anti-CD3-PerCP Cy5.5, anti-CD4-V500, anti-IFN-γ-PE, anti-IL-2-APC, anti-TNF-Alexa700, anti-CD44-V450 (BD Pharmingen), anti-CD8-PE Texas Red (Invitrogen), anti-IL-17 PE-Cy7, anti-IL-4-A488, and anti-IL-13-A488 (eBioscience), in Perm/Wash buffer 1× (BD Biosciences) for 20 min at RT, washed twice in Perm/Wash buffer, suspended in PBS. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell responses were analyzed using FlowJo software (TreeStar) applying the gating strategy described in Figure S1 in Supplementary Material.

Splenocytes from immunized mice were collected at indicated time points, plated at 1 × 10⁶/well in 96-well plates, stimulated with EsxAB, Hla_H35L, FhuD2, and Csa1A (10 μg/ml each) at 37°C 5% CO₂ for 96 h and incubated for the last 24 h at 37°C 5% CO₂ in the presence of 10 μM of Click-it^® Edu (Invitrogen). After two washing steps with PBS, cells were stained with Live/Dead Aqua (Invitrogen), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT in the dark and stained with anti-CD4 Pacific Blue and anti-CD44 APC (BD Biosciences). Cells were fixed with Cytofix (BD Biosciences), Click-it^® Edu detected following manufacturer’s instruction. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell proliferation was analyzed using FlowJo software (TreeStar) applying the gating strategy described in Figure S1 in Supplementary Material.

Splenocytes were isolated 12 days after vaccination, plated at 1-2x10⁶ cells/well in 96-well plates, and stimulated with vaccine proteins (Hla_H35L, EsxAB, FhuD2 and Csa1A, 10 μg/ml each) together with anti-CD28 and anti-CD49d mAb (2 μg/ml each, BD Biosciences) at 37°C for 16–18 h. Brefeldin A (5 μg/ml) was added for the last 4 h. The cells were then stained with Live/DeadYellow (Invitrogen), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT, and stained with the following fluorochrome-conjugated mAbs anti: CD3-PerCP Cy5.5, CD4-V500, IFN-γ-PE, IL-2-APC, TNF-AF700, CD44-V450 (BD Pharmingen), CD8-PE Texas Red (Invitrogen), IL-17A-PE Cy7, IL-4-AF488 and IL-13-AF488 (eBioscience) in Perm/Wash buffer (BD Biosciences) for 20 min at RT, washed twice in Perm/Wash buffer, suspended in PBS. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell responses were analyzed using FlowJo software (TreeStar) applying the gating strategy described in S1 Fig.