Pc7 cd34

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).

The ALDEFLUOR assay (StemCell Technologies) was used for the detection of ALDH expression in fresh and post-thaw CB HSCs. ALDH activity was measured by the protocol recommended by the manufacturer. Briefly, cell suspensions were adjusted to 10⁶ cells/mL with 1500 µL of ALDEFLUOR assay buffer after red blood cell depletion. ALDEFLUOR reagent (10 µL) was added to each tube, followed by 5 min of centrifugation at 300×g. Supernatants were stained with FITC-ALDH, APC A-750-CD38, phycocyanin (PC) 7-CD34, chrome orange-CD45 (Beckman Coulter), PE-CD73, and PC 5-CD90 (Becton Dickinson) antibodies and analyzed by flow cytometry (Beckman Coulter FC500). Diethylaminobenzaldehyde reagent was used to suppress ALDH activity in control tubes. Using the ALDH activity assay, CB HSCs were categorized as ALDH⁺ and ALDH^-.