Cytoflex b5 r3 v5

Transduced HSPCs were sorted based on expression of GFP or Cerulean. In vitro differentiation was analyzed on day 14 of culture. Flow cytometry analyses was performed on a Navios 10/3 or a CytoFLEX B5-R3-V5 (both Beckman Coulter). Kaluza 1.3/1.5 (Beckman Coulter) or FlowJo V10 were used for data analysis. Staining and measurement were performed according to standard protocols as previously described for human cells^{44 (link)}, using the antibodies FITC-CD8 (B9.11), FITC-CD19 (89B), FITC-CD38 (T16), FITC-CD41 (P2), FITC-CD66b (80H3), PE-CD42b (SZ2), PE-CD56 (IM2073U), PE-CD123 (9F5), PE-CD117 (95C3), PC5.5-CD14 (RMO52), PC7-CD3 (UCHT1), PC7-CD34 (581), PC7-CD41 (P2), PC7-CD235a (11E4B-7-6), APC-CD4 (13B8.2), APC-CD13 (Immu103.44), APC-CD34 (581), APC-CD45RA (2H4LDH11LDB9), AlexaFluor750-CD19 (89B), AlexaFluor750-CD235a (11E4B-7-6), KromOrange-CD3 (UCHT1) (all Beckman Coulter), PE-CD36 (CB38), PC7-CD66b (G10F5), and APC-CD42b (HIP1) (Becton Dickinson).

Spleens were harvested from mice bearing 17-day-old 4T1 tumors or tumor-naïve mice. Following dissociation and red blood cell lysis (Pharm Lyse™, BD Biosciences, Franklin Lakes, NJ, USA), Ly6G+ cells were enriched from spleens of tumor-bearing mice and Ly6G+, CD4+, and CD8+ cells were enriched from spleens of tumor-naïve mice using biotinylated anti-Ly6G antibody (clone 1A8, BioLegend), anti-CD4+ (clone GK1.5, BioLegend), or anti-CD8+ (53-6.7, BioLegend) bound to magnetic MojoSort™ streptavidin beads (BioLegend). CD11b+Ly6C+Ly6G+ granulocytes, CD3+CD4+ T cells, or CD3+CD8+ T cells were >95% pure, based on flow cytometric analysis. CD4+ and CD8+ T cells were labelled with 300 nM CFSE (Thermo Fisher Scientific), neutralized with RPMI 1640 containing 10% FBS, and then resuspended in serum-free AIM V media (Gibco). In a 96-well plate, 3 × 10⁵ T cells were stimulated with anti-CD3/anti-CD28-coated beads (Dynabeads™, Gibco) and 10⁵ Ly6G+ cells, in AIM V media, were added to some wells. Cells were gently pelleted by centrifugation at 100× g for 30 s and incubated at 37 °C/5% CO₂ for 72 h. Cells were then stained with anti-CD3-APC, anti-CD4-APC/Fire750, and anti-CD8-BV785 and proliferating T cells acquired on CytoFLEX B5-R3-V5 (Beckman Coulter, Brea, CA, USA) flow cytometer and analyzed using Kaluza (Beckman Coulter) software.